Large-scale production of soluble hyaluronidase

ABSTRACT

Provided are methods for preparing culture medium that contains soluble hyaluronidases. The methods employ cells that contain a plurality of active copies of nucleic acid encoding the soluble hyaluronidase and a plurality of feedings and temperature changes, whereby the encoded soluble hyaluronidase is secreted into the cell culture medium.

RELATED APPLICATIONS

This application is a divisional of allowed U.S. application Ser. No.12/735,868, to David Baker and Louis Bookbinder, entitled “LARGE-SCALEPRODUCTION OF SOLUBLE HYALURONIDASE,” filed Nov. 9, 2010, now U.S. Pat.No. 8,187,855 which is the national stage of International ApplicationNo. PCT/US09/001,455, to David Baker and Louis Bookbinder, entitled“LARGE-SCALE PRODUCTION OF SOLUBLE HYALURONIDASE,” filed Mar. 6, 2009,which claims benefit of priority to U.S. Provisional Application Ser.No. 61/068,622, to David Baker and Louis Bookbinder, entitled“LARGE-SCALE PRODUCTION OF SOLUBLE HYALURONIDASE,” filed Mar. 6, 2008.

This application is related to U.S. application Ser. No. 11/238,171 toLouis Bookbinder, Anirban Kundu, Gregory I. Frost, Michael F. Haller,Gilbert A. Keller, and Tyler M. Dylan, entitled SOLUBLEGLYCOSAMINOGLYCANS AND METHODS OF PREPARING AND USING SOLUBLEGLYCOSAMINOGLYCANS, filed Sep. 27, 2005 and published as U.S.Publication No. 20060104968, which is a continuation-in-part of U.S.application Ser. No. 11/065,716 to Louis Bookbinder, Anirban Kundu,Gregory I. Frost, Michael F. Haller, Gilbert A. Keller, and Tyler M.Dylan, entitled SOLUBLE GLYCOSAMINOGLYCANS AND METHODS OF PREPARING ANDUSING SOLUBLE GLYCOSAMINOGLYCANS, filed Feb. 23, 2005 and published asU.S. Publication No. 20050260186, which is a continuation-in-part ofU.S. application Ser. No. 10/795,095 to Louis Bookbinder, Anirban Kunduand Gregory I. Frost, entitled SOLUBLE HYALURONIDASE GLYCOPROTEIN(SHASEGP), PROCESS FOR PREPARING THE SAME, USES AND PHARMACEUTICALCOMPOSITIONS COMPRISING THEREOF, filed Mar. 5, 2004 and published asU.S. Publication No. 20040268425. The subject matter of each of theabove-noted applications is incorporated by reference in its entirety.

FIELD OF THE INVENTION

Provided are methods for large-scale production of a recombinant humanprotein.

BACKGROUND

Hyaluronidases are a family of enzymes that degrade hyaluronic acid(also known as hyaluronan or hyaluronate), an essential component of theextracellular matrix and a major constituent of the interstitialbarrier. By catalyzing the hydrolysis of hyaluronic acid, hyaluronidaselowers the viscosity of hyaluronic acid, thereby increasing tissuepermeability. As such, hyaluronidases have been used, for example, as aspreading or dispersing agent in conjunction with other agents, drugsand proteins to enhance their dispersion and delivery. Hyaluronidasesalso have other therapeutic and cosmetic uses. Because of the increasinguse of hyaluronidases for therapeutic and cosmetic uses, there is a needfor large-scale quantities of purified hyaluronidase. Therefore, amongthe objects herein, it is an object to provide methods for theproduction and purification of hyaluronidases.

SUMMARY

Provided herein are methods for the production and purification ofsoluble hyaluronidases. In particular, provided herein are methods forthe production and purification of soluble rHuPH20. Also provided hereinare cell medium and harvested cell culture fluid that contain solublerHuPH20. The methods provided herein can be used to produce and purifyany quantity of soluble hyaluronidase, such as rHuPH20. For example, themethods and steps described herein are amendable for scale-up orscale-down, as would be apparent to one of skill in the art.

The methods provided herein can be used to produce and purifylarge-scale quantities of soluble rHuPH20. The methods provided hereinfor producing soluble rHuPH20 can include a) inoculating cell medium ina bioreactor with an inoculum of cells that encode soluble rHuPH20 toproduce a cell culture, wherein cells contain between 150 and 300 copiesof nucleic acid encoding soluble rHuPH20, the bioreactor contains atleast 100 liters of cell culture and about 10¹⁰-10¹¹ cells areinoculated per 100 liters cell culture and cells are cultured at a settemperature; b) feeding the cells with a first feed medium containingglucose, L-alanyl-L-glutamine, human insulin and yeast extract inamounts sufficient to increase cell growth and peak cell density, and toincrease soluble rHuPH20 synthesis, wherein the feed medium is added tothe culture at a volume of 4% of the cell culture volume; c) feeding thecells with a second feed medium containing glucose,L-alanyl-L-glutamine, yeast extract and sodium butyrate in amountssufficient to increase soluble rHuPH20 synthesis and induce cell cyclearrest, wherein the amount of L-alanyl-L-glutamine is decreased comparedto the amount of L-alanyl-L-glutamine in the second step, and the amountof yeast extract is increased compared to the amount of yeast extract instep b), and the feed medium is added to the culture at a volume of 4%of the cell culture volume and the temperature is lowered compared tothe temperature in the step a) to a temperature sufficient to increasecell cycle arrest, increase cell viability and stabilize the solublehyaluronidase; d) feeding the cells with a third feed medium containingglucose, L-alanyl-L-glutamine, yeast extract and sodium butyrate inamounts sufficient to increase soluble rHuPH20 synthesis and increasecell cycle arrest, wherein the feed medium is added to the culture at avolume of 4% of the cell culture volume, the amount ofL-alanyl-L-glutamine and glucose is decreased compared to the amount ofL-alanyl-L-glutamine and glucose in step c), and the amount of yeastextract and sodium butyrate is increased compared to the amount of yeastextract and sodium butyrate in step c), and the temperature is loweredcompared to the temperature in step c) to a temperature sufficient toincrease cell cycle arrest, increase cell viability and stabilize thesoluble hyaluronidase; e) feeding the cells with a fourth feed mediumcontaining glucose, L-alanyl-L-glutamine, yeast extract and sodiumbutyrate in amounts sufficient to increase soluble rHuPH20 synthesis andincrease cell cycle arrest, wherein the amount of L-alanyl-L-glutamineand glucose is decreased compared to the amount of L-alanyl-L-glutamineand glucose in step d), the amount of sodium butyrate is decreasedcompared to the amount of sodium butyrate in step d), the temperature islowered compared to the temperature in step d) to a temperaturesufficient to increase cell cycle arrest, increase cell viability andstabilize the soluble hyaluronidase and feed medium is added to theculture at a volume of 4% of the cell culture volume; f) culturing thecells until the viability drops below at least or about 50%; g)obtaining the harvest cell culture fluid; and h) the soluble rHuPH20 ispurified from the harvest culture fluid.

The harvest cell culture fluid can be filtered prior to purification. Insome examples, the temperature in step a) is 37° C., the temperature instep c) is 36.5° C., the temperature in step d) is 36° C. and thetemperature in step e) is 35.5° C. The soluble rHuPH20 purification canbe effected by column chromatography, such as beaded crosslinked agarosecolumn chromatography, beaded crosslinked phenyl-substituted agarosecolumn chromatography, amino phenyl boronate column chromatography andhydroxyapatite column chromatography.

In one example, the method for producing soluble rHuPH20, includes stepsof a) inoculating cell medium in a bioreactor with an inoculum of cellsthat encode soluble rHuPH20 to produce a cell culture, wherein the cellscontain between 150 and 300 copies of nucleic acid encoding solublerHuPH20, the bioreactor contains at least 100 liters of cell culture,the inoculation cell density is at or about 4×10⁵ cells/mL and the cellsare cultured at 37° C.; b) feeding the cells with a first feed mediumcontaining at or about 33 g/L glucose, 32 mM L-alanyl-L-glutamine, 16.6g/L yeast extract and 33 mg/L insulin, wherein the feed medium is addedto the culture at a volume of 4% of the cell culture volume; c) feedingthe cells with a second feed medium containing at or about 33 g/Lglucose, 16 mM L-alanyl-L-glutamine, 33.4 g/L yeast extract and 0.92 g/Lsodium butyrate, wherein the feed medium is added to the culture at avolume of 4% of the cell culture volume and the temperature is loweredto 36.5° C.; d) feeding the cells with a third feed medium containing ator about 50 g/L glucose, 10 mM L-alanyl-L-glutamine, 50 g/L yeastextract and 1.8 g/L sodium butyrate, wherein the feed medium is added tothe culture at a volume of 4% of the cell culture volume and thetemperature is lowered to 36° C.; e) feeding the cells with a fourthfeed medium containing at or about 33 g/L glucose, 6.6 mML-alanyl-L-glutamine, 50 g/L yeast extract and 0.92 g/L sodium butyrate,wherein the feed medium is added to the culture at a volume of 4% of thecell culture volume and the temperature is lowered to 35.5° C.; f)continuing to culture the cells until viability drops below at least orabout 50%; g) obtaining the harvest cell culture fluid; h) filtering theharvest cell culture fluid; and i) purifying the rHuPH20 from theharvest culture fluid using beaded crosslinked agarose columnchromatography, beaded crosslinked phenyl-substituted agarose columnchromatography, amino phenyl boronate column chromatography andhydroxyapatite column chromatography.

In another example, the method for producing soluble rHuPH20 includesthe steps of a) inoculating cell medium in a bioreactor with an inoculumof cells that encode soluble rHuPH20 to produce a cell culture, whereinthe cells comprise between 150 and 300 copies of nucleic acid encodingsoluble rHuPH20; the bioreactor contains at least 100 liters of cellculture; the inoculation cell density is at or about 4×10⁵ cells/mL; andthe cells are cultured at or about 37° C.; b) feeding the cells with afirst feed medium containing or containing about 33 g/L glucose, 32 mML-alanyl-L-glutamine, 83.3 g/L yeast extract and 33 mg/L insulin,wherein the feed medium is added to the culture at a volume of 4% orabout 4% of the cell culture volume; c) feeding the cells with a secondfeed medium containing or containing about 33 g/L glucose, 13 mML-alanyl-L-glutamine, 166.7 g/L yeast extract and 0.92 g/L sodiumbutyrate, wherein the feed medium is added to the culture at a volume ofat or about 4% of the cell culture volume and the temperature is loweredto 36.5° C.; d) feeding the cells with a third feed medium containing orcontaining about 50 g/L glucose, 10 mM L-alanyl-L-glutamine, 250 g/Lyeast extract and 1.8 g/L sodium butyrate, wherein the feed medium isadded to the culture at a volume of 4% or about 4% of the cell culturevolume and the temperature is lowered to 36° C.; e) feeding the cellswith a fourth feed medium containing or containing about 33 g/L glucose,6.7 mM L-alanyl-L-glutamine, 250 g/L yeast extract and 0.92 g/L sodiumbutyrate, wherein the feed medium is added to the culture at a volume of4% or about 4% of the cell culture volume and the temperature is loweredto 35.5° C.; f) continuing to culture the cells until viability dropsbelow at least or about 50%; g) obtaining the harvest cell culturefluid; h) filtering the harvest cell culture fluid; and i) purifying therHuPH20 from the harvest culture fluid using beaded crosslinked agarosecolumn chromatography, beaded crosslinked phenyl-substituted agarosecolumn chromatography, amino phenyl boronate column chromatography andhydroxyapatite column chromatography.

In some examples, the volume of cell culture in the bioreactor is or isabout 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000 or 3500 liters.In some examples, the amount of soluble rHuPH20 is produced per 100 L ofcell culture using the methods provided herein at least or about 1, 5,10, 15, 20, 25, 30, 35 or 40 grams of soluble rHuPH20. The specificactivity of the soluble rHuPH20 can be at least or about 80000;100000,120000, 140000, 160000 or 180,000 units/mg. The cells that encodesoluble rHuPH20 can, in some instances, be DG44 CHO cells. Further, therHuPH20 can be encoded by nucleic acid set forth in SEQ ID NO:47.

Provided herein are cell culture media containing soluble rHuPH20 withan enzymatic activity of greater than 5000 units/mL, such as 10,000,12,000, 14,000, 16,000, 18,000, 20,000, 22,000 or 24,000 units/mL. Alsoprovided herein are harvested cell culture fluid containing solublerHuPH20 with an enzymatic activity of greater than 5000 units/mL, suchas 10,000, 12,000, 14,000, 16,000, 18,000, 20,000, 22,000 or 24,000units/mL.

DETAILED DESCRIPTION

-   Outline    -   A. Definitions    -   B. Overview    -   C. Hyaluronidase        -   1. Structure and function        -   2. PH20        -   3. Therapeutic uses of hyaluronidases            -   a. Use as a spreading agent            -   b. Use in hypodermoclysis            -   c. Use in vitrectomy and ophthalmic disorders and                conditions            -   d. Use in gene therapy            -   e. Cosmetic uses            -   f. Use in organ transplantation            -   g. Use in cancer treatment            -   h. Use in treatment of glycosaminoglycan accumulation in                the brain            -   i. Use in treatment of glycosaminoglycan accumulation in                cardiovascular disease            -   j. Use in pulmonary disease            -   k. Other uses    -   D. Hyaluronidase-expressing cells        -   a. 3D35M cells        -   b 2B2 cells    -   E. Cell culture expansion    -   F. Protein production    -   G. Protein concentration and buffer exchange    -   H. Purification        -   1. Beaded crosslinked agarose column        -   2. Beaded crosslinked phenyl-substituted agarose column        -   3. Amino Phenyl Boronate column        -   4. Hydroxyapatite column        -   6. Virus removal, protein concentration and buffer exchange    -   I. Filling    -   J. Monitoring and assays        -   1. Monitoring conditions        -   2. Monitoring soluble rHuPH20 production    -   K. Examples

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, Genbank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, hyaluronidase refers to an enzyme that degradeshyaluronic acid. Hyaluronidases include bacterial hyaluronidases (EC4.2.99.1), hyaluronidases from leeches, other parasites, and crustaceans(EC 3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35).Hyaluronidases also include any of non-human origin including, but notlimited to, murine, canine, feline, leporine, avian, bovine, ovine,porcine, equine, piscine, ranine, bacterial, and any from leeches, otherparasites, and crustaceans. Exemplary non-human hyaluronidases include,hyaluronidases from cows (SEQ ID NO:10), yellow jacket wasp (SEQ IDNOS:11 and 12), honey bee (SEQ ID NO:13), white-face hornet (SEQ IDNO:14), paper wasp (SEQ ID NO:15), mouse (SEQ ID NOS:16-18, 29), pig(SEQ ID NOS:19-20), rat (SEQ ID NOS:21-23, 28), rabbit (SEQ ID NO:24),sheep (SEQ ID NO:25), orangutan (SEQ ID NO:26), cynomolgus monkey (SEQID NO:27), guinea pig (SEQ ID NO:30), Staphylococcus aureus (SEQ IDNO:31), Streptococcus pyogenes (SEQ ID NO:32), and Clostridiumperfringens (SEQ ID NO:33). Exemplary human hyaluronidases include HYAL1(SEQ ID NO:34), HYAL2 (SEQ ID NO:35), HYAL3 (SEQ ID NO:36), HYAL4 (SEQID NO:37), and PH20 (SEQ ID NO:1). Also included amongst hyaluronidasesare soluble human PH20 and soluble rHuPH20 .

Reference to hyaluronidases includes precursor hyaluronidasepolypeptides and mature hyaluronidase polypeptides (such as those inwhich a signal sequence has been removed), truncated forms thereof thathave activity, and includes allelic variants and species variants,variants encoded by splice variants, and other variants, includingpolypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to theprecursor polypeptides set forth in SEQ ID NOS: 1 and 10-37, or themature form thereof. For example, reference to hyaluronidase alsoincludes the human PH20 precursor polypeptide variants set forth in SEQID NOS:48-49. Hyaluronidases also include those that contain chemical orposttranslational modifications and those that do not contain chemicalor posttranslational modifications. Such modifications include, but arenot limited to, pegylation, albumination, glycosylation, farnesylation,carboxylation, hydroxylation, phosphorylation, and other polypeptidemodifications known in the art.

As used herein, soluble human PH20 or sHuPH20 include maturepolypeptides lacking all or a portion of the glycosylphospatidylinositol (GPI) attachment site at the C-terminus such that uponexpression, the polypeptides are soluble. Exemplary sHuPH20 polypeptidesinclude mature polypeptides having an amino acid sequence set forth inany one of SEQ ID NOS:4-9 and 45-46. The precursor polypeptides for suchexemplary sHuPH20 polypeptides include a signal sequence. Exemplary ofthe precursors are those set forth in SEQ ID NOS:3 and 38-44, each ofwhich contains a 35 amino acid signal sequence at amino acid positions1-35. Soluble HuPH20 polypeptides also include those degraded during orafter the production and purification methods described herein.

As used herein, soluble rHuPH20 refers to a soluble form of human PH20that is recombinantly expressed in Chinese Hamster Ovary (CHO) cells.Soluble rHuPH20 is encoded by nucleic acid that includes the signalsequence and is set forth in SEQ ID NO:47. Also included are DNAmolecules that are allelic variants thereof and other soluble variants.The nucleic acid encoding soluble rHuPH20 is expressed in CHO cellswhich secrete the mature polypeptide. As produced in the culture mediumthere is heterogeneity at the C-terminus so that the product includes amixture of species that can include one or more of SEQ ID NOS. 4-9 invarious abundance. Corresponding allelic variants and other variantsalso are included, including those corresponding to the precursor humanPH20 polypeptides set forth in SEQ ID NOS:48-49. Other variants can have60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with any of SEQ ID NOS. 4 as long they retain ahyaluronidase activity and are soluble.

As used herein, “soluble rHuPH20-expressing cells” refers to any CHOcell that expresses soluble rHuPH20. Exemplary solublerHuPH20-expressing cells include 2B2 and 3D35M cells. SolublerHuPH20-expressing cells are CHO cells into which nucleic acid thatcontains the sequence set forth in SEQ ID NO:55 has been introduced.

As used herein, hyaluronidase activity refers to any activity exhibitedby a hyaluronidase polypeptide. Such activities can be tested in vitroand/or in vivo and include, but are not limited to, enzymatic activity,such as to effect cleavage of hyaluronic acid, ability to act as adispersing or spreading agent and antigenicity. hyaluronidase activityrefers to any activity exhibited by a hyaluronidase polypeptide.

As used herein, enzymatic activity refers to the activity of ahyaluronidase, as assessed in in vitro enzymatic assays, to cleave asubstrate, such as hyaluronic acid. In vitro assays to determine theenzymatic activity of hyaluronidases, such as soluble rHuPH20, are knowin the art and described herein. Exemplary assays include themicroturbidity assay described below (see e.g. Example 9 and section I)that measures cleavage of hyaluronic acid by hyaluronidase indirectly bydetecting the insoluble precipitate formed when the uncleaved hyaluronicacid binds with serum albumin.

As use herein, specific activity with reference to soluble rHuPH20 isthe enzyme activity relative to the amount of soluble rHuPH20, Specificactivity is calculated by dividing the enzymatic activity (units/mL) bythe protein concentration (mg/mL).

As used herein, “exhibits at least one activity” or “retains at leastone activity” refers to the activity exhibited by a variant solublerHuPH20 as compared to any soluble rHuPH20 set forth in SEQ ID NOS:4-9under the same conditions. Typically, a variant soluble rHuPH20 thatretains or exhibits at least one activity of a soluble rHuPH20 set forthin SEQ ID NOS:4-9 retains at or about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more of theactivity of a soluble rHuPH20 set forth in SEQ ID NOS:4-9. Exemplaryactivities include, but are not limited to, hyaluronidase activity andenzymatic activity.

As used herein, beaded crosslinked agarose column chromatography refersto chromatography using a column packed with beaded crosslinked agarose.Exemplary of beaded crosslinked agarose is Q Sepharose™.

As used herein, beaded crosslinked phenyl-substituted agarose columnchromatography refers to chromatography using a column packed withbeaded phenyl-substituted crosslinked agarose. Exemplary of beadedphenyl-substituted crosslinked agarose is Phenyl Sepharose™.

As used herein, amino phenyl boronate column chromatography refers tochromatography using a column packed with amino phenyl boronate agarose.

As used herein, hydroxyapatite column chromatography refers tochromatography using a column packed with hydroxyapatite.

As used herein, harvested cell culture fluid or harvest cell culturefluid (HCCF) refers to the fluid obtained following harvest of the cellsfrom the bioreactor and separation of the cell culture medium from thecells, cell debris and other aggregates. The cell culture that isharvested from the bioreactor can be filtered to clarify the culture,removing the cells, cell debris and other aggregates to leave theharvested cell culture fluid.

As used herein, cell density refers to the number of cells in a givenvolume of medium.

As used herein, cell culture or culture refers to a cell population thatis suspended in a medium under conditions suitable to maintain viabilityof the cells or grow the cells.

As used herein, medium, cell medium or cell culture medium refers to asolution containing nutrients sufficient to promote the growth of cellsin a culture. Typically, these solutions contain essential andnon-essential amino acids, vitamins, energy sources, lipids and/or traceelements. The medium also can contain additional supplements, such ashormones, growth factors and growth inhibitors. Reference to cellculture medium included

As used herein, the residues of naturally occurring α-amino acids arethe residues of those 20 α-amino acids found in nature which areincorporated into protein by the specific recognition of the chargedtRNA molecule with its cognate mRNA codon in humans.

As used herein, “in amounts sufficient to increase” when referring to asubstance increasing parameters such as cell growth rate, peak celldensity, protein synthesis or cell cycle arrest refers to the amount ofa substance that effects an increase in one of these parameters comparedto that observed in the absence of the substance. The parameters can beassessed in the presence and absence of a substance, and the amount ofsubstance that increases the parameter (such as cell growth rate, peakcell density, protein synthesis or cell cycle arrest) compared to in theabsence of the substance can be determined. The growth rate, peak celldensity, protein synthesis or cell cycle arrest in the presence of thesubstance can be increased by at or about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or morecompared to the growth rate, peak cell density, protein synthesis orcell cycle arrest in the absence of the substance.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules arecontemplated. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleicacids long.

As used herein, a peptide refers to a polypeptide that is from 2 to 40amino acids in length.

As used herein, the amino acids which occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 1). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain).

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3552-3559 (1969), andadopted 37 C.F.R. □§§1.821-1.822, abbreviations for amino acid residuesare shown in Table 1:

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp Aspartic acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence (Table 1) and modified andunusual amino acids, such as those referred to in 37 C.F.R.§§1.821-1.822, and incorporated herein by reference. Furthermore, itshould be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues, to an amino-terminal group such as NH₂ orto a carboxyl-terminal group such as COOH.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides.

As used herein, “non-natural amino acid” refers to an organic compoundthat has a structure similar to a natural amino acid but has beenmodified structurally to mimic the structure and reactivity of a naturalamino acid. Non-naturally occurring amino acids thus include, forexample, amino acids or analogs of amino acids other than the 20naturally-occurring amino acids and include, but are not limited to, theD-isostereomers of amino acids. Exemplary non-natural amino acids aredescribed herein and are known to those of skill in the art.

As used herein, a DNA construct is a single or double stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the sequence of amino acids of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity and/or homology of sequences ofresidues and the residues contained therein. Methods for assessing thedegree of similarity between proteins or nucleic acids are known tothose of skill in the art. For example, in one method of assessingsequence similarity, two amino acid or nucleotide sequences are alignedin a manner that yields a maximal level of identity between thesequences. “Identity” refers to the extent to which the amino acid ornucleotide sequences are invariant. Alignment of amino acid sequences,and to some extent nucleotide sequences, also can take into accountconservative differences and/or frequent substitutions in amino acids(or nucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

“Identity” per se has an art-recognized meaning and can be calculatedusing published techniques. (See, e.g.: Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exists a number of methodsto measure identity between two polynucleotide or polypeptides, the term“identity” is well known to skilled artisans (Carillo, H. & Lipton, D.,SIAM J Applied Math 48:1073 (1988)).

As used herein, homologous (with respect to nucleic acid and/or aminoacid sequences) means about greater than or equal to 25% sequencehomology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%,80%, 85%, 90% or 95% sequence homology; the precise percentage can bespecified if necessary. For purposes herein the terms “homology” and“identity” are often used interchangeably, unless otherwise indicated.In general, for determination of the percentage homology or identity,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; Carillo et al. (1988) SIAM J Applied Math 48:1073). By sequencehomology, the number of conserved amino acids is determined by standardalignment algorithms programs, and can be used with default gappenalties established by each supplier. Substantially homologous nucleicacid molecules would hybridize typically at moderate stringency or athigh stringency all along the length of the nucleic acid of interest.Also contemplated are nucleic acid molecules that contain degeneratecodons in place of codons in the hybridizing nucleic acid molecule.

Whether any two molecules have nucleotide sequences or amino acidsequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% “identical” or “homologous” can be determined using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)); Guide toHuge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994,and Carillo et al. (1988) SIAM J Applied Math 48:1073). For example, theBLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wis.). Percent homology or identity ofproteins and/or nucleic acid molecules can be determined, for example,by comparing sequence information using a GAP computer program (e.g.,Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith andWaterman (1981) Adv. Appl. Math. 2:482). Briefly, the GAP programdefines similarity as the number of aligned symbols (i.e., nucleotidesor amino acids), which are similar, divided by the total number ofsymbols in the shorter of the two sequences. Default parameters for theGAP program can include: (1) a unary comparison matrix (containing avalue of 1 for identities and 0 for non-identities) and the weightedcomparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, asdescribed by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE ANDSTRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979);(2) a penalty of 3.0 for each gap and an additional 0.10 penalty foreach symbol in each gap; and (3) no penalty for end gaps.

Therefore, as used herein, the term “identity” or “homology” representsa comparison between a test and a reference polypeptide orpolynucleotide. As used herein, the term at least “90% identical to”refers to percent identities from 90 to 99.99 relative to the referencenucleic acid or amino acid sequence of the polypeptide. Identity at alevel of 90% or more is indicative of the fact that, assuming forexemplification purposes a test and reference polypeptide length of 100amino acids are compared. No more than 10% (i.e., 10 out of 100) of theamino acids in the test polypeptide differs from that of the referencepolypeptide. Similar comparisons can be made between test and referencepolynucleotides. Such differences can be represented as point mutationsrandomly distributed over the entire length of a polypeptide or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g. 10/100 amino acid difference (approximately 90%identity). Differences are defined as nucleic acid or amino acidsubstitutions, insertions or deletions. At the level of homologies oridentities above about 85-90%, the result should be independent of theprogram and gap parameters set; such high levels of identity can beassessed readily, often by manual alignment without relying on software.

As used herein, an aligned sequence refers to the use of homology(similarity and/or identity) to align corresponding positions in asequence of nucleotides or amino acids. Typically, two or more sequencesthat are related by 50% or more identity are aligned. An aligned set ofsequences refers to 2 or more sequences that are aligned atcorresponding positions and can include aligning sequences derived fromRNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

As used herein, “primer” refers to a nucleic acid molecule that can actas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and a polymerization agent, such as DNApolymerase, RNA polymerase or reverse transcriptase) in an appropriatebuffer and at a suitable temperature. It will be appreciated that acertain nucleic acid molecules can serve as a “probe” and as a “primer.”A primer, however, has a 3′ hydroxyl group for extension. A primer canbe used in a variety of methods, including, for example, polymerasechain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR,multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′RACE, in situ PCR, ligation-mediated PCR and other amplificationprotocols.

As used herein, an allelic variant or allelic variation references anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wildtype form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species typicallyhave at least 80%, 90% or greater amino acid identity with a wildtypeand/or predominant form from the same species; the degree of identitydepends upon the gene and whether comparison is interspecies orintraspecies. Generally, intraspecies allelic variants have at leastabout 80%, 85%, 90% or 95% identity or greater with a wildtype and/orpredominant form, including 96%, 97%, 98%, 99% or greater identity witha wildtype and/or predominant form of a polypeptide. Reference to anallelic variant herein generally refers to variations in proteins amongmembers of the same species.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude substitutions, deletions and insertions of nucleotides. Anallele of a gene also can be a form of a gene containing a mutation.

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, modification is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids and nucleotides, respectively. Methods ofmodifying a polypeptide are routine to those of skill in the art, suchas by using recombinant DNA methodologies.

As used herein, the term promoter means a portion of a gene containingDNA sequences that provide for the binding of RNA polymerase andinitiation of transcription. Promoter sequences are commonly, but notalways, found in the 5′ non-coding region of genes.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

The term substantially free of cellular material includes preparationsof proteins in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly-produced. In oneembodiment, the term substantially free of cellular material includespreparations of enzyme proteins having less that about 30% (by dryweight) of non-enzyme proteins (also referred to herein as acontaminating protein), generally less than about 20% of non-enzymeproteins or 10% of non-enzyme proteins or less that about 5% ofnon-enzyme proteins. When the enzyme protein is recombinantly produced,it also is substantially free of culture medium, i.e., culture mediumrepresents less than about or at 20%, 10% or 5% of the volume of theenzyme protein preparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of enzyme proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of enzyme proteins having less than about 30% (by dryweight) 20%, 10%, 5% or less of chemical precursors or non-enzymechemicals or components.

As used herein, synthetic, with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, an expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses that are operatively linked toexogenous genes to transfer (as vehicles or shuttles) the exogenousgenes into cells.

As used herein the term assessing is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of a protease, or a domain thereof, present inthe sample, and also of obtaining an index, ratio, percentage, visual orother value indicative of the level of the activity. Assessment can bedirect or indirect and the chemical species actually detected need notof course be the proteolysis product itself but can for example be aderivative thereof or some further substance. For example, detection ofa cleavage product of a complement protein, such as by SDS-PAGE andprotein staining with Coomasie blue.

As used herein, a composition refers to any mixture. It can be asolution, suspension, liquid, powder, paste, aqueous, non-aqueous or anycombination thereof.

As used herein, a kit is a packaged combination that optionally includesother elements, such as additional reagents and instructions for use ofthe combination or elements thereof.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treatment encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease. Treatment also encompasses any pharmaceuticaluse of a modified interferon and compositions provided herein.

As used herein, a pharmaceutically effective agent, includes anytherapeutic agent or bioactive agents, including, but not limited to,for example, anesthetics, vasoconstrictors, dispersing agents,conventional therapeutic drugs, including small molecule drugs andtherapeutic proteins.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein, a patient refers to a human subject.

As used herein, an effective amount is the quantity of a therapeuticagent necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease or disorder.

As used herein, animal includes any animal, such as, but are not limitedto primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer, sheep; ovine, such as pigs and other animals. Non-human animalsexclude humans as the contemplated animal. The hyaluronidases providedherein are from any source, animal, plant, prokaryotic and fungal. Mostenzymes are of animal origin, including mammalian origin.

As used herein, a control refers to a sample that is substantiallyidentical to the test sample, except that it is not treated with a testparameter, or, if it is a plasma sample, it can be from a normalvolunteer not affected with the condition of interest. A control alsocan be an internal control.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a compound, comprising “an extracellular domain”includes compounds with one or a plurality of extracellular domains.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 mM” means “about 5 mM” and also “5 mM.”

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or IUPAC-IUB Commission onBiochemical Nomenclature (see, (1972) Biochem. 11:1726).

B. Overview

Provided herein are methods for the large scale production of solublehyaluronidases, such as soluble human hyaluronidases, including solublehuman PH20 (sHuPH20), such as, for example, soluble rHuPH20. The methodstypically utilize bioreactors to culture cells that produce the solublehyaluronidase, such as CHO cells (e.g. DG44 CHO cells). Exemplary ofsuch cells are 2B2 cells, which produce soluble rHuPH20. The volume ofcell culture in the bioreactor can range from 1 L to 5000 L or more, buttypically is or is about 200, 300, 400, 500, 1000, 1500, 2000, 2500,3000 or 3500 liters. Prior to inoculation of the bioreactor, the cellsare expanded through a series of increasing cell culture volumes togenerate the required number of cells for seeding of the bioreactor.Typically, the cell culture in the bioreactor is seeded with 10⁵ to 10⁶cells/mL, but can be seeded with more or less. The cells are thenincubated in the bioreactor for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or more days.

During this incubation, feed media is added to the cell culture tosupply additional nutrients and supplements. Exemplary supplements ornutrients that can be included in the feed media include, but are notlimited to, glucose, glutamine or glutamine-substitute, such asL-alanyl-L-glutamine, insulin, and sodium butyrate. The type and amountof supplement added can influence cell growth and protein production.For example, insulin and glutamine or glutamine-substitute can beincorporated into the first feed media added to the cell culture toincrease cell growth and peak cell density. Subsequent feed media can bedesigned to promote protein production more than cell growth.Supplements such as insulin can be excluded or reduced in amount, as canglutamine or glutamine-substitute, such as L-alanyl-L-glutamine. Incontrast, supplements such as yeast extract that enhance proteinsynthesis can be increased in amount. In addition, supplements thatenhance cell cycle arrest and, therefore, increased protein production,also can be included. Exemplary of such supplements is sodium butyrate.

Following protein production in the bioreactor, the cells are harvestedand the soluble hyaluronidase, such as soluble rHuPH20, that has beensecreted into the cell culture media is concentrated prior to initiationof the purification process. The soluble hyaluronidase is then purifiedfrom the concentrated protein solution using a series of purificationsteps. Exemplary of purification methods that are used for the methodsherein is a combination of ion-exchange chromatography, hydrophobicinteraction chromatography and affinity chromatography. The purifiedprotein is then concentrated and diafiltered.

Utilizing the methods described herein, between about 0.5-50 grams ofsoluble hyaluronidases, such as soluble rHuPH20, is produced per 100 Lof cell culture. In some examples, the amount of soluble rHuPH20produced per 100 L of culture is or is about 1, 2, 3, 4, 5, 10, 15, 20,30, or 40 grams or more. In some examples, the yield of solublehyaluronidase following purification can range from between or betweenabout 10% to 50% of the amount produced before purification. Forexample, the yield following purification can be or be about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% or 50% of the amount produced beforepurification. Generally, the specific activity of soluble rHuPH20produced using the methods herein is at least or about 80000, 100000,120000, 140000, 160000 or 180,000 units/mg.

C. Hyaluronidases

Hyaluronidases are a family of enzymes that degrade hyaluronic acid(also known as hyaluronan or hyaluronate), an essential component of theextracellular matrix and a major constituent of the interstitialbarrier. By catalyzing the hydrolysis of hyaluronic acid, hyaluronidaselowers the viscosity of hyaluronic acid, thereby increasing tissuepermeability. As such, hyaluronidases have been used, for example, as aspreading or dispersing agent in conjunction with other agents, drugsand proteins to enhance their dispersion and delivery.

1. Structure and function of hyaluronidases

There are three general classes of hyaluronidases; mammalianhyaluronidase, bacterial hyaluronidase and hyaluronidase from leeches,other parasites and crustaceans. Mammalian-type hyaluronidases (EC3.2.1.35) are endo-β-N-acetyl-hexosaminidases that hydrolyze the β1→4glycosidic bond of hyaluronic acid into various oligosaccharide lengthssuch as tetrasaccharides and hexasaccharides. They have both hydrolyticand transglycosidase activities and can degrade hyaluronic acid andchondroitin sulfates, such as C4-S and C6-S. Hyaluronidases of this typeinclude, but are not limited to, hyaluronidases from cows (SEQ IDNO:10), yellow jacket wasp (SEQ ID NOS:11 and 12), honey bee (SEQ IDNO:13), white-face hornet (SEQ ID NO:14), paper wasp (SEQ ID NO:15),mouse (SEQ ID NOS:16-18, 29), pig (SEQ ID NOS:19-20), rat (SEQ IDNOS:21-23, 228), rabbit (SEQ ID NO:24), sheep (SEQ ID NO:25), orangutan(SEQ ID NO:26), cynomolgus monkey (SEQ ID NO:27), guinea pig (SEQ IDNO:30) and human hyaluronidases.

There are six hyaluronidase-like genes in the human genome: HYAL1,HYAL2, HYAL3, HYAL4, HYALP1 and PH20/SPAM 1. HYALP1 is a pseudogene, andHYAL3 (SEQ ID NO:36) has not been shown to possess enzyme activitytoward any known substrates. HYAL4 (precursor polypeptide set forth inSEQ ID NO:37) is a chondroitinase and exhibits little activity towardshyaluronic acid. HYAL1 (precursor polypeptide set forth in SEQ ID NO:34)is the prototypical acid-active enzyme and PH20 (precursor polypeptideset forth in SEQ ID NO:1) is the prototypical neutral-active enzyme.Acid-active hyaluronidases, such as HYAL1 and HYAL2 (precursorpolypeptide set forth in SEQ ID NO:35) generally lack catalytic activityat neutral pH (i.e. pH 7). For example, HYAL1 has little catalyticactivity in vitro over pH 4.5 (Frost et al. (1997) Anal Biochemistry251:263-269). HYAL2 is an acid-active enzyme with a very low specificactivity in vitro. The hyaluronidase-like enzymes also can becharacterized by those which are generally locked to the plasma membranevia a glycosylphosphatidyl inositol anchor such as human HYAL2 and humanPH20 (Danilkovitch-Miagkova, et al. (2003) Proc Natl Acad Sci USA.100(8):4580-5), and those which are generally soluble such as humanHYAL1 (Frost et al, (1997) Biochem Biophys Res Commun. 236(1):10-5). Bycatalyzing the hydrolysis of hyaluronic acid, a major constituent of theinterstitial barrier, hyaluronidase lowers the viscosity of hyaluronicacid, thereby increasing tissue permeability. It also has been shown toexhibit anti-cancer and anti-carcinogenic activities.

N-linked glycosylation of some hyaluronidases can be very important fortheir catalytic activity and stability. While altering the type ofglycan modifying a glycoprotein can have dramatic affects on a protein'santigenicity, structural folding, solubility, and stability, manyenzymes are not thought to require glycosylation for optimal enzymeactivity. Hyaluronidases are, therefore, unique in this regard, in thatremoval of N-linked glycosylation can result in near completeinactivation of the hyaluronidase activity. For such hyaluronidases, thepresence of N-linked glycans is critical for generating an activeenzyme.

There are seven potential N-linked glycosylation sites at N82, N166,N235, N254, N368, N393, N490 of human PH20 exemplified in SEQ ID NO: 1.Disulfide bonds form between the cysteine residues C60 and C351 andbetween C224 and C238 to form the core hyaluronidase domain. However,additional cysteines are required in the carboxy terminus for neutralenzyme catalytic activity such that amino acids 36 to 464 of SEQ ID NO:1contains the minimally active human PH20 hyaluronidase domain. Thus,N-linked glycosylation site N490 is not required for properhyaluronidase activity.

N-linked oligosaccharides fall into several major types (oligomannose,complex, hybrid, sulfated), all of which have (Man)3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn residuesthat fall within-Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro).Glycosylation at an-Asn-Xaa-Cys-site has been reported for coagulationprotein C. In some instances, the hyaluronidase can contain bothN-glycosidic and O-glycosidic linkages. For example, rHuPH20 (asproduced in the methods described herein) has O-linked oligosaccharidesas well as N-linked oligosaccharides.

The methods described herein provide a process for the production andpurification of large quantities of a soluble preparation of human PH20hyaluronidase preparation.

2. PH20

Human PH20 (also known as sperm surface protein PH20), as noted above,is the prototypical neutral-active enzyme that is generally locked tothe plasma membrane via a glycosylphosphatidyl inositol (GPI) anchor. Itis naturally involved in sperm-egg adhesion and aids penetration bysperm of the layer of cumulus cells by digesting hyaluronic acid. ThePH20 mRNA transcript is normally translated to generate a 509 amino acidprecursor protein containing a 35 amino acid signal sequence at theN-terminus (amino acid residue positions 1-35). The mature PH20polypeptide is, therefore, a 474 amino acid polypeptide with an aminoacid sequence set forth in SEQ ID NO:2.

Soluble forms of human PH20 (sHuPH20) can be produced and purified usingthe methods described herein. The generation of sHuPH20 are described inrelated U.S. patent application Ser. Nos. 10/795,095, 11/065,716 and11/238,171 (also referred to in these applications as sHASEGP orrHuPH20), and in Examples 1 and 4, below. The soluble forms are producedby expressing nucleic acid encoding C-terminal truncations of the maturePH20 polypeptide that lack the GPI-attachment sites. Soluble forms ofhuman PH20 include soluble rHuPH20, which is produced and purified usingthe methods provided herein.

3. Therapeutic Uses of Hyaluronidases

Various forms of hyaluronidases have been prepared and approved fortherapeutic use in humans. For example, animal-derived hyaluronidasepreparations include Vitrase® (ISTA Pharmaceuticals), a purified ovinetesticular hyaluronidase, and Amphadase® (Amphastar Pharmaceuticals), abovine testicular hyaluronidase. Hylenex® (Halozyme Therapeutics) is ahuman recombinant hyaluronidase produced by genetically engineeredChinese Hamster Ovary (CHO) cells containing nucleic acid encoding forsoluble rHuPH20. Approved therapeutic uses for hyaluronidase include useas an adjuvant to increase the absorption and dispersion of otherinjected drugs, for hypodermoclysis (subcutaneous fluid administration),and as an adjunct in subcutaneous urography for improving resorption ofradiopaque agents. In addition to these indications, hyaluronidases,including sHuPH20, can be used as a therapeutic or cosmetic agent forthe treatment of additional diseases and conditions.

As noted above, hyaluronidase is a spreading or diffusing substancewhich modifies the permeability of connective tissue through thehydrolysis of hyaluronic acid, a polysaccharide found in theintercellular ground substance of connective tissue, and of certainspecialized tissues, such as the umbilical cord and vitreous humor. Whenno spreading factor is present, materials injected subcutaneously, suchas drugs, proteins, peptides and nucleic acid, spread very slowly.Co-injection with hyaluronidase, however, can cause rapid spreading. Therate of diffusion is proportional to the amount of enzyme, and theextent of diffusion is proportional to the volume of solution.Absorption and dispersion of injected drugs and agents can be enhancedby adding 10-1000 units hyaluronidase to the injection solution. In someexamples, 150 U hyaluronidase is added. Hyaluronidases have multipleuses, including and in addition to their use as a spreading agent.Hyaluronidase is commonly used, for example, for peribulbar block inlocal anesthesia prior ophthalmic surgery. The presence of the enzymeprevents the need for additional blocks and speeds the time to the onsetof akinesia (loss of eye movement). Peribulbar and sub-Tenon's block arethe most common applications of hyaluronidase for ophthalmic procedures.Hyaluronidase also can promote akinesia in cosmetic surgery, such asblepharoplasties and face lifts. Exemplary therapeutic and cosmetic usesfor hyaluronidase are described below.

a. Use as a Spreading Agent

Hyaluronidases, such as soluble rHuPH20 produced using the methodsdescribed herein, can be used to promote or enhance the delivery agentsand molecules to any of a variety of mammalian tissues in vivo. It canbe used to facilitate the diffusion and, therefore, promote thedelivery, of small molecule pharmacologic agents as well as largermolecule pharmacologic agents, such as proteins, nucleic acids andribonucleic acids, and macromolecular compositions than can contain acombination of components including, but not limited to, nucleic acids,proteins, carbohydrates, lipids, lipid-based molecules and drugs. Forexample, molecules and macromolecular complexes ranging from about 10 nmto about 500 nm in diameter, can exhibit dramatic improvements indelivery through interstitial spaces when the interstitial space hasbeen previously, or is coincidentally, exposed to hyaluronidase (seee.g. U.S. patent application Ser. Nos. 10/795,095, 11/065,716 and11/238,171).

Examples of pharmaceutical, therapeutic and cosmetic agents andmolecules that can be administered with hyaluronidase include, but arenot limited to, anesthetics; anti-metabolites, anti-neoplastics andother anti-cancer agents; anti-virals; anti-infectives, includinganti-bacterials and other antibiotics, anti-fungals and otheranti-infectives; immunomodulatory agents; steroidal and non-steroidalanti-inflammatories; beta blockers; sympathomimetics; ducosanoids,prostaglandins and prostaglandin analogs; miotics, cholinergics andanti-cholinesterases; anti-allergenics and decongestants; hormonalagents; growth factors; immunosuppressants; vaccines and toxoids; immunesera; antibodies; and any combination thereof. In one example, solublerHuPH20 is administered with a cathepsin, such as cathepsin L.

b. Use in Hypodermoclysis

Hypodermoclysis, the infusion of fluids and electrolytes into thehypodermis of the skin, is a useful and simple hydration techniquesuitable for mildly to moderately dehydrated adult patients, especiallythe elderly. Although considered safe and effective, the most frequentadverse effect is mild subcutaneous edema that can be treated by localmassage or systemic diuretics. Approximately 3 L can be given in a24-hour period at two separate sites. Common infusion sites include thechest, abdomen, thighs and upper arms. Solutions used in hypodermoclysisinclude, for example, normal saline, half-normal saline, glucose withsaline and 5% glucose. Potassium chloride also can be added to thesolution. The addition of hyaluronidase to the solution can enhancefluid absorption and increase the overall rate of administration.

c. Use in Vitrectomy and Ophthalmic Disorders and Conditions

Hyaluronidase can be used to minimize the detachment or tearing of theretina during vitrectomy. This could cause, for example, the vitreousbody to become uncoupled or “disinserted” from the retina, prior toremoval of the vitreous body. Such disinsertion or uncoupling of thevitreous body can minimize the likelihood that further tearing ordetachment of the retina will occur as the vitreous body is removed.

Hyaluronidase can be used for various ophthalmic applications, includingthe vitrectomy adjunct application described in U.S. Pat. No. 5,292,509.The use of a highly purified hyaluronidase, such as, for example,soluble rHuPH20 produced and purified by the methods described herein,is preferable for intraocular procedures to minimize immunogenicity andtoxicity. In some examples, a pegylated hyaluronidase can be used toprolong residence within the vitreous and prevent localized uptake.

Hyaluronidases can be used to treat and/or prevent ophthalmic disordersby, for example, preventing neovascularization and increasing the rateof clearance from the vitreous of materials toxic to the retina.Hyaluronidase can be administered in an amount effective to liquefy thevitreous humor of the eye without causing toxic damage to the eye.Liquefaction of the vitreous humor increases the rate of liquid exchangefrom the vitreal chamber. This increase in exchange removes thecontaminating materials whose presence can cause ophthalmologic andretinal damage.

Hyaluronidase also can be used to reduce postoperative pressure.Hyaluronic acid has been used in eye primarily as a spacer duringcataract and intraocular lens surgical procedures. It also is used inother ocular surgical procedures such as glaucoma, vitreous and retinasurgery and in corneal transplantation. A common side effect occurringin postoperative cataract patients is a significant early, andoccasionally prolonged, rise in intraocular pressure. Such a conditionis sometimes serious, especially in patients with glaucomatous opticdisc changes. Hyaluronidase can be co-administered with hyaluronic acidto the eye prior to surgery to reduce postoperative pressure in the eye.The hyaluronidase is administered in an amount effective to reduce theintraocular pressure to pre-operative levels by breaking down thehyaluronic acid without decreasing its effectiveness during surgery norcausing side effects in the patient (U.S. Pat. No. 6,745,776).

Hyaluronidase also can be administered to patients with glaucoma toremove glycosaminoglycans from the trabecular meshwork and reduceintraocular pressure, and can be applied to the vitreous to promote theresolution of vitreous hemorrhages (i.e. extravasation of blood into thevitreous), which can occur in connection with conditions such asdiabetic retinopathy, retinal neovascularization, retinal veinocclusion, posterior vitreous detachment, retinal tears, ocular traumasand the like. The presence of vitreous hemorrhages, which are typicallyslow to resolve, can delay, complicate or prevent procedures thatrequire the retina to be visualized through the vitreous for diagnosisand/or for treatment procedures such as laser photocoagulation and thelike which are often primary treatments for conditions such asproliferative diabetic retinopathy.

d. Use in Gene Therapy

The efficacy of most gene delivery vehicles in vivo does not correspondto the efficacy found observed in vitro. Glycosaminoglycans can hinderthe transfer and diffusion of DNA and viral vectors into many celltypes. The levels such extracellular matrix material can hinder theprocess considerably. Administration of hyaluronidase can open channelsin the extracellular matrix, thus enhancing delivery of gene therapy.For example, hyaluronidase can be administered with collagenase tofacilitate transduction of DNA in vivo (Dubensky et al. (1984) Proc NatlAcad Sci USA 81(23):7529-33). Hyaluronidase also can enhance genetherapy using adeno-associated virus (Favre et al, (2000) Gene Therapy7(16):1417-20). The channels opened following administration ofhyaluronidase are of a size that typically enhance diffusion of smallermolecules such as retroviruses, adenoviruses, adeno-associated virusesand DNA complexes (as well as other therapeutic and pharmacologicalagents of interest). The pores are not so large, however, as to promotethe dislocation and movement of cells.

In some examples, viruses can be engineered to express hyaluronidase tofacilitate their replication and spread within a target tissue. Thetarget tissue can be, for example, a cancerous tissue whereby the virusis capable of selective replication within the tumor. The virus also canbe a non-lytic virus wherein the virus selectively replicates under atissue specific promoter. As the viruses replicate, the co-expression ofhyaluronidase with viral genes can facilitate the spread of the virus invivo.

e. Cosmetic Uses

Hyaluronidases can be by administered to remove glycosaminoglycansinvolved in the accumulation of cellulite and to promote lymphatic flow.In some examples, human hyaluronidases, such as for example, solublerHuPH20, are used for the treatment of cellulite. The hyaluronidase canbe administered through repeated subcutaneous injections, throughtransdermal delivery in the form of ointments or creams or through theuse of injectable slow release formulations to promote the continualdegradation of glycosaminoglycans and prevent their return.

Hyaluronidase also can be used to treat conditions such as “pigskin”edema or “orange peel” edema. Hyaluronidases can effect depolymerizationof the long mucopolysaccharide chains that can accumulate in the dermisand which are responsible for the retention of bound water and of theslowing, by capillary compression, of the diffusion of organic liquids,which eliminate metabolic wastes. Such retention of water and wastesassociated with fat overloading of the lipocytes, constitutes classical“pigskin” edema or “orange peel” edema. Depolymerization can cut thelong chains of mucopolysaccharides into shorter chains, resulting in theelimination of the bound water and wastes and restoration of the venousand lymphatic circulation, culminating in the disappearance of localedema.

f. Use in Organ Transplantation

The content of hyaluronic acid in an organ can increase withinflammation. An increased concentration of hyaluronic acid has beenobserved in tissue from different organs characterized byinflammatory-immunological injury such as alveolitis (Nettelbladt et al.(1991) Am. Rev. Resp. Dis. 139: 759-762) and myocardial infarction(Waldenstrom et al. (1991) J. Clin. Invest. 88(5): 1622-1628). Otherexamples include allograft rejection after a renal (Ha'llgren et al.(1990) J. Exp. Med. 171: 2063-2076; Wells et al. (1990) Transplantation50: 240-243), small bowel (Wallander et al. (1993) Transplant. Int. 6:133-137) or cardiac (Haellgren et al. (1990) J Clin Invest 185:668-673)transplantation; or a myocardial inflammation of viral origin(Waldenstrdm et al. (1993) Eur. J. Clin. Invest. 23: 277-282). Theoccurrence of interstitial edemas in connection with the grafting of anorgan constitutes a severe problem in the field of transplantationsurgery. Grafts with interstitial edemas can swell to such a degree thatthe function is temporarily be lost. In some instances, the swelling cancause disruption of the kidney, resulting in a massive hemorrhage.Hyaluronidases can be used to degrade accumulated glycosaminoglycans inan organ transplant. Removal of such glycosaminoglycans promotes removalof water from the graft and thus enhances organ function.

g. Use in Cancer Treatment

Hyaluronidase has direct anticarcinogenic effects. Hyaluronidaseprevents growth of tumors transplanted into mice (De Maeyer et al.,(1992) Int. J. Cancer 51:657-660) and inhibits tumor formation uponexposure to carcinogens (Pawlowski et al. (1979) Int. J. Cancer23:105-109) Hyaluronidase is effective as the sole therapeutic agent inthe treatment of brain cancer (gliomas) (WO 198802261). In addition tothese effects, hyaluronidases also can be used to enhance penetration ofchemotherapeutic agents into solid tumors. They can be injectedintratumorally with anti-cancer agents or intravenously for disseminatedcancers or hard to reach tumors. The anticancer agent can be achemotherapeutic, an antibody, a peptide, or a gene therapy vector,virus or DNA. Additionally, hyaluronidase can be used to recruit tumorcells into the cycling pool for sensitization in previouslychemorefractory tumors that have acquired multiple drug resistance (StCroix et al., (1998) Cancer Lett September 131(1): 35-44).Hyaluronidases, such as, for example soluble rHuPH20, also can enhancedelivery of biologics such as monoclonal antibodies, cytokines and otherdrugs to tumors that accumulate glycosaminoglycans.

Hyaluronidases can also be used to increase the sensitivity of tumorsthat are resistant to conventional chemotherapy. For example,hyaluronidase, such as soluble rHuPH20, can be administered to a patienthaving a tumor associated with a HYAL1 defect in an amount effective toincrease diffusion around the tumor site (e.g., to facilitatecirculation and/or concentrations of chemotherapeutic agents in andaround the tumor site), inhibit tumor cell motility, such as byhyaluronic acid degradation, and/or to lower the tumor cell apoptosisthreshold. This can bring the tumor cell(s) to a state of anoikis, whichrenders the tumor cell more susceptible to the action ofchemotherapeutic agents. Administration of hyaluronidase can induceresponsiveness of previously chemotherapy-resistant tumors of thepancreas, stomach, colon, ovaries, and breast (Baumgartner et al. (1988)Reg. Cancer Treat. 1:55-58; Zanker et al. (1986) Proc. Amer. Assoc.Cancer Res. 27:390).

In one example, hyaluronidases are used in the treatment of metastaticand non-metastatic cancers, including those that have decreasedendogenous hyaluronidase activity relative to non-cancerous cells.Hyaluronidases can be used as a chemotherapeutic agent alone or incombination with other chemotherapeutics. Exemplary cancers include, butare not limited to, small lung cell carcinoma, squamous lung cellcarcinoma, and cancers of the breast, ovaries, head and neck, or anyother cancer associated with depressed levels of hyaluronidase activityor decreased hyaluronic acid catabolism.

h. Use in Treatment of Glycosaminoglycan Accumulation in the Brain

Hyaluronic acid levels are elevated in a number of cerebrospinalpathologic conditions. Levels of cerebrospinal hyaluronic acid arenormally less than 200 μg/L in adults (Laurent et al. (1996) Acta NeurolScand September 94(3):194-206), but can elevate to levels of over 8000μg/L in diseases such as meningitis, spinal stenosis, head injury andcerebral infarction. Hyaluronidases, such as, for example, solublerHuPH20, can be utilized to degrade critically elevated levels ofsubstrate.

The lack of effective lymphatics in the brain also can lead to lifethreatening edema following head trauma. Hyaluronic acid accumulation isa result of increased synthesis by hyaluronic acid synthases anddecreased degradation. Accumulation of hyaluronic acid can initiallyserve the beneficial purpose of increasing water content in the damagedtissue to facilitate leukocyte extravasation, but continued accumulationcan be lethal. Administration of hyaluronidase, such as intrathecally orintravenously, to a patient suffering from head trauma can serve toremove tissue hyaluronic acid accumulation and the water associated withit.

Hyaluronidases also can be used in the treatment of edema associatedwith brain tumors, particularly that associated with glioblastomamultiform. The edema associated with brain tumors results from theaccumulation of hyaluronic acid in the non-cancerous portions of thebrain adjacent the tumor. Administration of hyaluronidase to the sitesof hyaluronic acid accumulation (e.g., by intravenous injection or via ashunt) can relieve the edema associated with such malignancies bydegrading the excess hyaluronic acid at these sites.

i. Use in Treatment of Glycosaminoglycan Accumulation in CardiovascularDisease

Hyaluronidase can be used in the treatment of some cardiovasculardisease. Administration of hyaluronidase in animal models followingexperimental myocardial infarct can reduce infarct size (Maclean, et al(1976) Science 194(4261):199-200). One proposed mechanism by which thiscan occur is by reducing hyaluronic acid accumulation that occursfollowing ischemia reperfusion. Reduction of infarct size is believed tooccur from increased lymph drainage and increased tissue oxygenation andreduction of myocardial water content.

Hyaluronidases also can be used to limit coronary plaques fromarteriosclerosis. Such plaques accumulate glycosaminoglycans and mediatemacrophage and foam cell adhesion (Kolodgie et al. (2002) ArteriosclerThromb Vasc Biol. 22(10):1642-8).

j. Use in Pulmonary Disease

Levels of hyaluronic acid in broncheoalveolar lavages (BAL) from normalindividuals are generally below 15 ng/ml. However, hyaluronic acidlevels in BAL rise dramatically in conditions of respiratory distress(Bjermer et al. (1987) Br Med J (Clin Res Ed) 295(6602):803-6). Theincreased hyaluronic acid in the lung can prevent oxygen diffusion andgas exchange as well as activating neutrophil and macrophage responses.Purified preparations of soluble rHuPH20, such as those produced usingthe methods provided herein, can be delivered by either pulmonary orintravenous delivery to patients presenting with such conditions toreduce hyaluronan levels. Hyaluronidases also can be administered topatients suffering from other pulmonary complications that areassociated with elevated glycosaminoglycans or to enhance the deliveryof other co delivered molecules to the lung.

k. Other Uses

In further examples of its therapeutic use, hyaluronidase can be usedfor such purposes as an antidote to local necrosis from paravenousinjection of necrotic substances such as vinka alkaloids (Few et al.(1987) Amer. J. Matern. Child Nurs. 12, 23-26), treatment of ganglioncysts (Paul et al. (1997) J Hand Surg. 22 (2): 219-21) and treatment oftissue necrosis due to venous insufficiency (Elder et al. (1980) Lancet648-649). Hyaluronidases also can be used to treat ganglion cysts (alsoknown as a wrist cyst, Bible cyst, or dorsal tendon cyst), which are themost common soft tissue mass of the hand and are fluid filled sacs thatcan be felt below the skin.

Hyaluronidases can be used in the treatment of spinal cord injury bydegrading chondroitin sulfate proteoglycans (CSPGs). Following spinalcord injury, glial scars containing CSPGs are produced by astrocytes.CSPGs play a crucial role in the inhibition of axon growth. In addition,the expression of CSPG has been shown to increase following injury ofthe central nervous system (CNS). Hyaluronidases also can be utilizedfor the treatment of herniated disks in a process known aschemonucleolysis. Chondroitinase ABC, an enzyme cleaving similarsubstrates as hyaluronidase, can induce the reduction of intradiscalpressure in the lumbar spine. There are three types of disk injuries. Aprotruded disk is one that is intact but bulging. In an extruded disk,the fibrous wrapper has torn and the NP has oozed out, but is stillconnected to the disk. In a sequestered disk, a fragment of the NP hasbroken loose from the disk and is free in the spinal canal.Chemonucleolysis is typically effective on protruded and extruded disks,but not on sequestered disk injuries.

D. Soluble rHuPH20-Expressing Cells

The methods described herein can be used to generate and purify largequantities of soluble rHuPH20, Soluble rHuPH20 is expressed in CHO cellsthat are grown in large-scale cell culture. Expression is effected usingan expression vector that contains the nucleotide sequence encoding thesequence of amino acids set forth in SEQ ID NO:3 (corresponding to aminoacids 1 to 482 of the precursor human PH20 polypeptide set forth in SEQID NO:1). Following translation, the 35 amino acid signal sequence iscleaved and soluble rHuPH20 is secreted into the medium. The vector alsocontains an IRES downstream of the soluble rHuPH20 encoding region, amouse dihydrofolate reductase gene and the SV40 pA sequence. Theexpression vector was introduced into DG44 cells, which aredihydrofolate reductase deficient (dhfr-) that have been adapted to growin suspension culture in a chemically defined, animal product-freemedium. The resulting soluble rHuPH20-expressing cells include thosedescribed in Examples 1 and 4, below, and include cells designated3D35M, 2B2, 3E10B, 1B3, 5C1, 1G11 and 2G10 cells.

Other cells can be used to produce hyaluronidases similar to rHuPH20.Generally, protein expression systems suitable for the introduction ofcritical N-linked glycosylation residues on hyaluronidases are used.Such cells include, for example, yeast cells, fungal cells, plant cells,insect cells and mammalian cells. Many cell lines are available formammalian expression including mouse, rat human, monkey, chicken andhamster cells. Exemplary cell lines include but are not limited to CHO(including DG44 cells and CHO-S cells), Balb/3T3, HeLa, MT2, mouse NSO(nonsecreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are availableadapted to serum-free media, which facilitates purification of secretedproteins from the cell culture media.

a. 3D35M Cells

Exemplary of soluble rHuPH20-expressing cells are 3D35M cells, describedin Example 1, below, and U.S. Patent Publication Nos. 20040268425,20050260186 and 20060104968. 3D35M cells are dihydrofolate reductasedeficient (dhfr-) DG44 CHO cells that express soluble rHuPH20. The cellswere transformed with an HZ24 expression vector having the nucleotidesequence set forth in SEQ ID NO:50. This vector contains a CMV promoterdriving expression of nucleic acid encoding a 482 amino acid (SEQ IDNO:3) polypeptide that corresponds to amino acid positions 1 to 482 ofthe full length human PH20 set forth in SEQ ID NO:1. This includes a 35amino acid N-terminal signal sequence. The vector also contains aninternal ribosome entry site (IRES) after the PH20-encoding sequence,followed by a mouse dihydrofolate reductase gene and the SV40polyadenylation sequence. Following translation, the 482 amino acidpolypeptide is processed to remove the 35 amino acid signal sequence,resulting in the secretion of soluble rHuPH20.

Characterization of 3D35M cells demonstrated that the nucleic acidregion encoding soluble rHuPH20 is present in the cells at a copy numberof approximately 318 copies/cells. Soluble rHuPH20 produced from 3D35Mcells by the methods herein is a mixture of species that can include oneor more of the polypeptides having sequences set forth in SEQ IDNOS:4-9. In an exemplary characterization of these species (described inExample 11), the species set forth in SEQ ID NO:4 was present at anabundance of 0.2%, the species set forth in SEQ ID NO:5 (correspondingto amino acids 1 to 446 of SEQ ID NO:4) was present at an abundance of18.4%, the species set forth in SEQ ID NO:6 (corresponding to aminoacids 1 to 445 of SEQ ID NO:4) was present at an abundance of 11.8%, thespecies set forth in SEQ ID NO:7 (corresponding to amino acids 1 to 444of SEQ ID NO:4) was present at an abundance of 56.1%; and the speciesset forth in SEQ ID NO:8 (corresponding to amino acids 1 to 443 of SEQID NO:4) was present at an abundance of 13.6%. Such heterogeneity in thesoluble rHuPH20 preparation is likely a result of C-terminal cleavage bypeptidases present during the production and purification methodsprovided herein.

The 3D35M cells can be grown in cell culture medium with or withoutmethotrexate. Additional supplements, such as glutamine, also can beadded. In some examples, the cells are grown in cell culture mediumcontaining, for example, 50 nM, 100 nM, 500 nM, 1 or 2 μM methotrexateand lacking hypoxanthine and thymidine. In one example, 3D35M cells arecultured at 37° C. in 5-7% CO₂ in culture medium (such as CD CHO Medium,Invitrogen) without hypoxanthine and thymidine and with 100 nMmethotrexate and glutamine or a glutamine substitute, such asL-alanyl-L-glutamine, a stabilized, dipeptide form of L-glutamine. Othercell culture media appropriate for CHO cells can be used to culture3D35M cells including, but not limited to, Dulbecco's modified Eagle'smedium (DMEM), Eagle's Minimum essential medium (EMEM), Iscove'smodified Eagle's medium (IMEM), F12 and RPMI. 3D35M cells grown undersuch conditions in shaking flasks can produce in excess of 1000 units/mLhyaluronidase activity. When cultured in a bioreactor, such as describedin Example 3, below, 3D35M cells can produce soluble rHuPH20 withenzymatic activity in excess of 2000 units/mL.

b. 2B2 Cells

Exemplary of soluble rHuPH20-expressing cells for production of rHuPH20in the methods provided herein are described in Example 4 and designated2B2 cells. 2B2 cells were generated by adapting 3D35M cells to highermethotrexate levels (i.e. 20 μM) and selecting clones that grew in thehigher methotrexate concentration. This adaptation increased thehyaluronidase activity produced by the cells. DG44 cells aredihydrofolate reductase-deficient (dhfr-) and, therefore, cannot makenucleosides. The expression vector present in 3D35M and 2B2 cellscontains, in addition to the PH20 gene, the coding sequence for mousedihydrofolate reductase. Methotrexate is a strong competitivedihydrofolate reductase inhibitor. Therefore, by increasing theconcentration of methotrexate in the culture media, thehyaluronidase-expressing cells are forced to produce more mousedihydrofolate reductase to remain viable. This can be effected by, forexample, gene amplification or rearrangement of the integrated DNA to amore stable and productive arrangement. Thus, forcing an increase in theproduction of mouse dihydrofolate reductase also can result in anincrease in the production of sHuPH20. A comparison of enzymaticactivity of soluble rHuPH20 produced by 2B2 cells and 3D35M cellsdemonstrated that activity was typically between 80% and 100% higher in2B2 cells (see e.g. Example 5, below) compared to 3D35M cells.

2B2 cells were selected from amongst the cell clones that were isolatedfollowing selection with 20 μM methotrexate as the cell line thatproduced soluble rHuPH20 having the greatest enzymatic activity (see,e.g. Example 4). When characterized, it was observed that the nucleicacid region encoding soluble rHuPH20 was present in 2B2 cells at a copynumber of approximately 206 copies/cell. Southern blot analysis of SpeI-, Xba I- and BamH I/Hind III-digested genomic 2B2 cell DNA using aprobe specific for the nucleic acid region encoding soluble rHuPH20revealed the following restriction digest profile: one major hybridizingband of ˜7.7 kb and four minor hybridizing bands (˜13.9, ˜6.6, ˜5.7 and˜4.6 kb) with DNA digested with Spe I; one major hybridizing band of˜5.0 kb and two minor hybridizing bands (˜13.9 and ˜6.5 kb) with DNAdigested with Xba I; and one single hybridizing band of ˜1.4 kb observedusing 2B2 DNA digested with BamH UHind III.

2B2 cells can be grown in cell culture medium with or withoutmethotrexate. Additional supplements, such as glutamine, insulin andyeast extract also can be added. In some examples, the cells are grownin cell culture medium containing, for example, 50 nM, 100 nM, 500 nM, 1μM, 2 μM, 10 μM, 20 μM or more methotrexate and lacking hypoxanthine andthymidine. In one example, 2B2 cells are cultured at 37° C. in 5-7% CO₂in culture medium (such as CD CHO Medium, Invitrogen) withouthypoxanthine and thymidine and with 20 μM methotrexate and glutamine orL-alanyl-L-glutamine, a stabilized, dipeptide form of L-glutamine. Othercell culture media appropriate for CHO cells can be used to culture 2B2cells, including, but not limited to, Dulbecco's modified Eagle's medium(DMEM), Eagle's Minimum essential medium (EMEM), Iscove's modifiedEagle's medium (IMEM), F12 and RPMI. 2B2 cells grown under suchconditions in shaking flasks can produce in excess of 3000 units/mLhyaluronidase activity. When cultured in a bioreactor, such as describedin Example 8, below, 2B2 cells can produce soluble rHuPH20 havingenzymatic activity in excess of 17000 units/mL hyaluronidase activity.

Soluble rHuPH20 produced from 2B2 cells by the methods herein is amixture of species of polypeptides having sequences set forth in SEQ IDNOS:4-9. In an exemplary characterization the soluble rHuPH20 productproduced by 2B2 cells (described in Example 11), the species set forthin SEQ ID NO:4 was present at an abundance of 1.9%, the species setforth in SEQ ID NO:5 (corresponding to amino acids 1 to 446 of SEQ IDNO:4) was present at an abundance of 46.7%, the species set forth in SEQID NO:6 (corresponding to amino acids 1 to 445 of SEQ ID NO:4) waspresent at an abundance of 16.7%, the species set forth in SEQ ID NO:7(corresponding to amino acids 1 to 444 of SEQ ID NO:4) was present at anabundance of 27.8%; and the species set forth in SEQ ID NO:8(corresponding to amino acids 1 to 443 of SEQ ID NO:4) was present at anabundance of 6.9%. As noted for soluble rHuPH20 produced from 3D35Mcells, the heterogeneity in the soluble rHuPH20 preparation from 2B2cells is likely a result of C-terminal cleavage by peptidases presentduring the production and purification methods provided herein.

E. Cell Culture Expansion

The methods described herein employ bioreactors to grow large volumes ofcell culture to produce large quantities of soluble rHuPH20. Asdescribed in detail below, these methods include a cell expansion phase,a protein production phase, a protein concentration and buffer exchangephase, and a purification phase. The soluble rHuPH20-expressing cells,such as 2B2 cells, are initially expanded from an original inoculum,such as an aliquot of cells from a working cell bank (WCB) or mastercell bank (MCB), to a larger volume prior to culture in the bioreactorfor the production phase. The final culture volume in the expansionphase is directly proportional to the volume of the bioreactor used inthe following production phase. Typically, a larger bioreactor isinoculated using a larger final culture volume from the expansion phasethan is a smaller bioreactor.

The soluble rHuPH20-expressing cells are expanded through a series ofcultures, each increasing in volume from the previous one, and eachbeing used as the inoculum for the subsequent culture. Exemplary of suchcells are 2B2 cells. The original inoculum is typically one in which thepurity and identity of the cells and the cell number are defined. Thesecells can be stored frozen, such as at −20° C., −70° C. or −80° C., orcan be maintained in liquid media at, for example, 4° C., or maintainedin culture at, for example, 37° C. In some instances, the originalinoculum is a master cell bank or working cell bank aliquot that hasbeen stored frozen. In such cases, the inoculum is thawed, such as in a37° C. waterbath. The original cell inoculum is typically centrifugedand the cells are resuspended in an appropriate cell culture media. Forexample, 2B2 cells can be resuspended in, and subsequently cultured in,basal media, such as CD CHO media (Invitrogen), or reconstitutedpowdered CD CGO AGT™ media (Invitrogen), supplemented with 8 mMglutamine or L-alanyl-L-glutamine and 20 μM methotrexate. In anotherexample, cells can be grown in basal media supplemented with 8 mMglutamine or L-alanyl-L-glutamine and 100 mM methotrexate. Any othersuitable cell culture media also can be used to expandhyaluronidase-expressing cells. For example, cells can be cultured inDulbecco's modified Eagle's medium (DMEM), Eagle's Minimum essentialmedium (EMEM), Iscove's modified Eagle's medium (IMEM), F12, RPMI, orother chemically-defined or undefined media, with or without additionalsupplements. Typically, the cells are grown in serum-free media, butalso can be grown in media containing serum. One of skill in the artcould prepare cell culture media using other basal cell culture media towhich various nutrients can be supplemented to make the cell culturemedia in which soluble rHuPH20-expressing cells are cultured.

The cell inoculum is added to the first of a series of increasingvolumes of cell culture media, thus expanding the cell culture.Following the initial inoculation, the cells are expanded in ahumidified incubator or bioreactor at an appropriate temperature with anappropriate amount of CO₂. Typically, the amount of CO₂ is between 4%and 9%, typically between 6.0% and 8.0%, such as 7.0% and thetemperature is between 35° C. and 39° C., typically between 36° C. and38° C., such as 37° C. For example, 2B2 and 3D35M cells can be grown ina 37° C. humidified incubator with 7% CO₂. The culture can be agitated,such as at 90-130 rpm, during this process. When the cells reach thedesired density, such as, for example, greater than 1.0×10⁶ cells/mL(e.g. between 1.5×10⁶ cells/mL and 2.5×10⁶ cells/mL), the cell cultureis used to inoculate a larger volume of fresh cell culture media. Forexample, cells can be inoculated into the next culture at a density of4×10⁴ to 4×10⁶ cells/mL, typically 2×10⁵ to 6×10⁵ cells/mL, such as4×10⁵ cells/mL. The process is repeated until the cells have beenexpanded to the desired volume and cell density for seeding of, forexample, 4×10⁴ to 4×10⁶ cells/mL into the bioreactor.

In one example, soluble rHuPH20-expressing cells, such as 2B2 cells, areinitially added to approximately 20 mL of fresh cell culture media in a125 mL shaker flask, resulting in a culture volume of 20-30 mL,typically 25 mL. Following incubation at 37° C., 7% CO₂ and expansion ofthe cells to a density of greater than 1.5×10⁶ cells/mL, fresh media isadded to the flask to expand the cell culture to 40 mL. The cells areincubated again until a density of greater than 1.5×10⁶ cells/mL isattained, after which the entire cell culture (approximately 40 mL) isadded to fresh media to make 100 mL culture volume in a 125 mL spinnerflask. This process is repeated by transferring the entire cell culture(approximately 100 mL) to a 250 mL spinner flask containing sufficientfresh media to make a final culture volume of 200 mL, then a 1 L spinnerflask containing sufficient fresh media to make a final culture volumeof 800 mL, then a 6 L spinner flask containing sufficient fresh media tomake a final culture volume a final volume of 5 L, and finally to a 36 Lspinner flask containing sufficient fresh media to make a final culturevolume of 32 L. Between each transfer, the cells are incubated until theculture reached a density of greater than 1.5×10⁶ cells/mL. In someexamples, a higher cell density is attained following incubation of thefinal 36 L spinner flask. For example, cells in the 36 L spinner flaskcan be expanded to a cell density of 3.55×10⁶ cells/mL to 6.05×10⁶cells/mL. This process can be used to expand soluble rHuPH20-expressingcells before introduction to a 400 L bioreactor (300 L culture volume)for the protein production phase (see, e.g. Example 8). Smaller volumesof cell culture and different cell densities can be used for smallerbioreactors. For example, cells can be expanded to a volume ofapproximately 20 L with a cell density of between 1.8×10⁶ cells/mL and2.5×10⁶ cells/mL prior to introduction to a 125 L bioreactor. In anotherexample, soluble rHuPH20-expressing cells are expanded to a volume ofapproximately 800 mL with a cell density of between 1.5×10⁶ cells/mL and2.5×10⁶ cells/mL prior to introduction to a 5 L bioreactor.

This process, like any of the processes described herein, also can bescaled-up by one of skill in the art for introduction of the cells intoa bioreactor with a culture volume larger than 300 L. For example, theprocess can be scaled up for introduction of the cells into a bioreactorwith a 2500 L culture volume, such as described in Example 12. Thus, inone example of the methods provided herein, following thawing, the cellsare serially expanded through a 125 mL shaker flask (working volume of20-30 mL, such as 25 mL), a 250 mL shaker flask (working volume of 45-55mL, such as 50 mL), a 1 L shaker flask (working volume of 190-210 mL,such as 200 mL), two 2 L shaker flasks (working volume of 350-450 mL perflask, such as 400 mL per flask), six 2 L shaker flasks (working volumeof 350-450 mL per flask, such as 400 mL per flask), a 25 L wavebioreactor (working volume of 14-16 L, such as 15 L), a 100 L wavebioreactor (working volume of 75-85 L, such as 80 L), and a 600 L seedbioreactor (working volume of 440-520 L, such as 480 L).

F. Protein Production

Following cell expansion, the soluble rHuPH20-expressing cells aretransferred to a bioreactor for the production phase, during which largequantities of soluble rHuPH20 are secreted into the cell media. Thisphase typically is designed such that the cells growth is maximized inthe first half of the bioreactor run, and soluble rHuPH20 production ismaximized in the second half of the bioreactor run. The cells areprovided with a series of feed media at particular time pointsthroughout the production to regulate this process. The bioreactorconditions also are typically monitored to ensure optimal conditions aremaintained throughout the process. The methods described herein forprotein can be scaled up or down by one of skill in the art. Further,modifications to, for example, cell media, incubation times, feedingprotocols. One of skill in the art can empirically determine theappropriate conditions for protein production for any given bioreactorand cell type.

Bioreactors of different sizes and designs can be utilized in themethods herein. Bioreactors with working volumes of between 1 L and 5000L or more can be used in the methods herein. In some examples, a 5 L, 36L, 125 L, 400 L or 3500 L bioreactor is used in the methods herein toculture cells in volumes of approximately 4 L, 23 L, 100 L, 300 L and2500 L, respectively. Typically, the bioreactor is sterilized prior tothe addition of cell culture media or cells. Sterilization can beeffected by autoclaving or otherwise treating with steam for somebioreactors, or by treatment with a sterilizing solution, such as dilutesodium hydroxide, dilute nitric acid or sodium hypochlorite. In someexamples, the bioreactor is sterilized by steam at 121° C., 20 PSI for30 minutes. Following sterilization, cell culture media can be added tothe bioreactor and then assessed for contamination, such by microbialcontamination, after a period of time to ensure that the sterilizationprocess was effective.

The cell culture from the expansion phase, described above, is added tothe sterilized bioreactor containing fresh cell culture media.Generally, the soluble rHuPH20-expressing cells are inoculated into thefresh cell culture media at a cell density of 10⁴ to 10⁷ cells/mL, suchas 10⁵ to 10⁶ cells/mL. For example, cells can be inoculated at adensity of 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 4×10⁶, or 1×10⁷ cells/mL.In one example, soluble rHuPH20-expressing cells are inoculated at acell density of 4×10⁵ cells/mL. The total cell count followinginoculation can be, therefore, between 10⁷ and 10¹⁴, depending on thesize of the bioreactor and the cell density. For example, a cell culturevolume of 100 L can have a cell density following inoculation ofapproximately 10⁹, 10¹⁰, 10¹¹ or 10¹² cells. In another example, a cellculture volume of 2500 L can have a cell density following inoculationof approximately 10¹⁰, 10¹¹, 10¹² or 10¹³ cells.

The volumes of inoculating cell culture and fresh culture media used aredependent upon the size of the bioreactor and the cell density of theinoculum. For example, approximately 30 L of soluble rHuPH20-expressingcells, such as 2B2 cells, can be added to a 400 L bioreactor containing230 L fresh cell culture media, for a total volume of approximately 260L and an inoculation cell density of 4×10⁵ cells/mL (total cell count ofapproximately 10¹¹ cells). This can be scaled up or down as necessary,depending on the bioreactor. For example, approximately 20 L of solublerHuPH20-expressing cells, such as 3D35M cells, can be added to a 125 Lbioreactor containing 65 L fresh cell culture media, for a total volumeof approximately 85 L and an inoculation cell density of 4×10⁵ cells/mL(total cell count of approximately 3.4×10¹⁰ cells. In another example,for production in a 3500 L bioreactor, 2B2 cells are added to fresh cellculture media for a total cell culture volume of 1900-2300 L, such as2100 L.

The fresh cell culture media contains the appropriate supplements toprovide the necessary nutrients to the cells to promote cell growth.Supplements that can be added to the basal cell medium include, but arenot limited to, glucose, insulin, sodium butyrate, yeast extract andglutamine or a glutamine substitute, such as L-alanyl-L-glutamine. Insome instances, the basal medium contains sufficient glucose that nofurther glucose needs to be added. In other instances, glucose is addedto the media later in the production process, such as in a subsequentfeed media. The addition of insulin to the medium can promote cellgrowth and increase peak cell density. Glutamine orglutamine-substitutes, such as or L-alanyl-L-glutamine, can support cellcycle progression and also enhance cell growth. One of skill in the artcan empirically determine the amount and quality of the nutrients thatcan be supplemented to the basal medium. In some examples, glutamine orglutamine-substitute is added to the basal cell culture medium at 1 mM,2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM or 20 mM.Insulin can be added to the cell culture medium at, for example, 0.5mg/L to 50 mg/L, such as 1 mg/L to 40 mg/L, 2 mg to 30 mg/L, or 5 mg/Lto 20 mg/L. For example, basal cell culture medium supplemented with 5mg/L insulin and 8 mM L-alanyl-L-glutamine can be used as the fresh cellculture media into which the soluble rHuPH20-expressing cells areinoculated. Additional supplements, such as antibiotics, anti-fungals,indicators, salts, vitamins, amino acids and growth factors also can beadded.

The individual parameters of the bioreactor can be set to maintainoptimal conditions throughout the protein production process. Thespecific parameters that can be set depend on the bioreactor used, andcan include, but are not limited to, temperature, pH, dissolved oxygen,impeller speed, vessel pressure, air sparge and air overlay. In oneexample, the conditions of a 125 L bioreactor containing 100 L cellculture of 3D35M cells are set to; temperature: 37° C.; dissolvedoxygen: 25%±10%; impeller speed: 50 RPM; vessel pressure: 3 psi; airsparge: 1 L/minute; air overlay: 1 L/minute, pH:7.2 In another example,the conditions of a 400 L bioreactor containing an initial cell culturevolume of 260 L are set to; temperature: 37° C.; impeller speed 40-55RPM; vessel pressure: 3 psi; air sparge: 0.5-1.5 L/minute; air overlay:1 L/minute. In a further example, the conditions of a 3000 L bioreactorcontaining an initial culture volume of 2100 L are set to; temperature:37° C. (or between 36.5° C. and 37.5° C.; impeller speed: 35 RPM (or70-80 RPM); vessel pressure: 5 psi (or 3-7 psi); air sparge: 12 L/minute(or 11-13 L/minute); dissolved oxygen: 25%, or >25%; pH pre inoculation:7.2 (or pH 7.1-7.3); pH post inoculation: ≦7.2 (or ≦7.3). One of skillin the art can empirically determine the appropriate conditions forgrowth of a particular soluble rHuPH20-expressing cell in a particularbioreactor.

The soluble rHuPH20-expressing cells are typically cultured in thebioreactor for between 10 and 25 days. In some examples, the solublerHuPH20-expressing cells are cultured in the bioreactor for 12, 13, 14,15 or 16 days before harvesting. In other examples, the cells areharvested when the viable cell count (VCC) falls to a particular level,such as, for example, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 70%. Inone example, the cells are harvested when the VCC is between 30% and35%. In another example, the cells are harvested within 24 hours of theVCC dropping below 50%.

During the bioreactor culture, the cells can be grown as batch cultures,in which the culture is grown to completion without the addition offurther nutrients. In other examples, the cells are grown as fed-batchcultures and provided with a series of feed media at particular timepoints to supplement nutrients and glucose throughout. In someinstances, the nutrients provided in the cell culture medium into whichthe cells were inoculated have depleted by 3, 4, 5, 6, 7 days or morepost-inoculation. Thus, providing additional nutrients or supplementscan produce higher yields of protein than batch cultures. In oneexample, cells are provided with feed media on days 6, 9 and 11post-inoculation. In another example, cells are provided with feed mediaon days 7, 9 and 11 post-inoculation. In a further example, cells areprovided with feed media on days 5, 7, 9 and 11 post-inoculation. Thevolume of feed media added to the bioreactor culture can range between,for example, 0.5% and 20%, such as 1-20%, 2-15%, 3-10% or 4-5% of thecell culture volume. In some instances, the feed media is added at avolume equivalent to 4% of the cell culture volume.

The addition of various supplements to the feed media also can be usedto regulate the growth and/or cell cycle of the cells. Exemplarynutrients and supplements that can be included in the feed mediainclude, but are not limited to, glutamine or glutamine-substitute, suchas L-alanyl-L-glutamine, insulin, yeast extract, glucose and sodiumbutyrate or sodium butyrate. Furthermore, the basal media used in thefeed media also can be concentrated, thus providing additionalnutrients, such as essential amino acids, that may have been depletedduring cell culture. The basal media in the feed media can be 2×, 3×,4×, 5×, 6× or more concentrated. In other examples, the basal media isless concentrated, or the same concentration as the cell culture mediain the bioreactor.

The supplements included in the feed media can be used to regulate cellgrowth and protein production. For example, the first feed media addedto the cell culture can include nutrients that enhance cell cycleprogression, cell growth and peak cell density. Subsequent feed mediascan promote cell growth arrest and/or protein synthesis. The amount ofeach supplement in each feed media can vary, such as by increasing ordecreasing from one feed media to the next, or can be the same from onefeed media to the next. In some examples, the amount of a supplement inincreased from one feed media to the next, such as by 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or more. Inother examples, the amount of a supplement in decreased from one feedmedia to the next, such as by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more. In one example, a supplement is omitted from a feedmedia. In other examples, the amount of a supplement in the feed mediastays the same. One of skill in the art can empirically determine theoptimum amount of each supplement for each feed media to promote thedesired amount of cell growth and protein production.

Exemplary supplements or nutrients that can be included in the feedmedia include, but are not limited to, glucose, glutamine orglutamine-substitute, such as L-alanyl-L-glutamine, insulin, and sodiumbutyrate. The type and amount of supplement added can influence cellgrowth and protein production. For example, insulin and glutamine orglutamine-substitute can be incorporated into the first feed media addedto the cell culture to increase cell growth and peak cell density.Subsequent feed media can be designed to promote protein production morethan cell growth. Supplements such as insulin can be excluded or reducedin amount, as can glutamine or glutamine-substitute, such asL-alanyl-L-glutamine. In contrast, supplements such as yeast extractthat enhance protein synthesis can be increased in amount. In addition,supplements that enhance cell cycle arrest and, therefore, increasedprotein production, also can be included. Exemplary of such supplementsis sodium butyrate.

In one example, insulin is added to one or more feed media. The additionof insulin can increase peak cell density by, for example, 2%, 5%, 10%,15%, 20%, 25%, 30% or more. Although insulin can be incorporated intoany feed media, typically, insulin is added to early feed media, such asthe first feed media, or the first and second feed media, to promotemaximal cell growth in the initial phase of the bioreactor run. Forexample, a feed media, such as the first feed media, can contain amountof insulin at or about 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, 25 mg/L, 30mg/L, 35 mg/L, 40 mg/L, 45 mg/L, 50 mg/L, 55 mg/L, 60 mg/L or more. Incontrast, the amount of insulin added to later feed media can be reducedor can be completely omitted.

Glutamine or glutamine-substitute, such as L-alanyl-L-glutamine, alsocan be added to the feed media. In some instances, the amount ofglutamine or glutamine-substitute added to the first feed media is morethan the amount of glutamine or glutamine-substitute added to subsequentfeed media. In particular examples, the amount of glutamine orglutamine-substitute added to each subsequent feed media is reducedcompared to the amount added in the prior feed media. The optimal amountadded to each feed media can be determine empirically by one of skill inthe art, and can include, for example, concentrations of glutamine orglutamine-substitute at or about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM,30 mM, 35 mM, 40 mM, 45 mM, 50 mM or more.

Typically, the basal media used in the feed media also is supplementedwith glucose. The amount of glucose added to each feed media can beincreased or decreased relative to the previous feed media, or can stayapproximately constant. In some examples, the amount of glucose added tothe feed media is or is about 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35g/L, 40 g/L, 45 g/L, 50 g/L, 55 g/L, 60 g/L, 75 g/L, 80 g/L or more.

In addition, supplements that promote protein synthesis also can beincluded. Such nutrients include, for example, yeast extract. In someinstances, the amount of yeast extract included in the feed media isincreased during the bioreactor run. For example, the amount of yeastextract in the third feed media can be increased compared to the amountin the second feed media, which can be increased compared to the amountin the second feed media. In some examples, the amount of yeast extractadded to the feed media is between 5 and 1000 g/L, such as or as about10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 75 g/L, 100 g/L, 125 g/L, 150g/L, 175 g/L, 200 g/L, 250 g/L, 300 g/L, 350 g/L, 400 g/L or more.

Supplements that enhance cell cycle arrest and, therefore, increaseprotein production, also can be included. Typically, such supplementsare included in feed media that are added to the bioreactor later in therun and not included in the first feed media. For example, supplementsthat enhance cell cycle arrest can be added to the second feed media andsubsequent feed media. Exemplary of such supplements is sodium butyrate.In some examples, the amount of sodium butyrate added to the feed mediais between 0.1 g/L and 10 g/L, such as or as about 0.2 g/L, 0.3 g/L, 0.4g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1.0 g/L, 1.1 g/L, 1.2g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6 g/L, 1.7 g/L, 1.8 g/L, 1.9 g/L, 2.0g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L, or more.

Further, any one or more of the bioreactor conditions can be alteredduring the production phase to optimize protein production. In oneexample, the temperature is lowered. This can serve to promote cellcycle arrest, prolong cell viability (thereby increasing total proteinproduction), and help stabilize the hyaluronidase that has beensecreted. For example, the temperature of the bioreactor can be reducedat each feeding, such as from 37° C. to 36.5° C. on the second feeding,to 36° C. on the third feeding and to 35.5° C. on the fourth feeding.One of skill in the art can empirically determine the appropriate feedmedia and the time at which to provide the feed, as well as theappropriate conditions in the bioreactor.

In one example, cells are provided with feed media on days 6, 9 and 11post-inoculation. In another example, cells are provided with feed mediaon days 7, 9 and 11 post-inoculation. In a further example, cells areprovided with feed media on days 5, 7, 9 and 11 post-inoculation. Thefeed media provided at each time-point can be the same or different, andcan include supplements such as, but not limited to, glucose, sodiumbutyrate, insulin, glutamine or a glutamine substitute and yeastextract. For example, 2B2 cells growing in a 260 L culture in a 400 Lbioreactor can be provided with a first feed at day 5 containing 10.4 Lof 4× basal media (e.g. CD CHO media) with 33 g/L Glucose, 32 mML-alanyl-L-glutamine, 16.6 g/L Yeast extract and 33 mg/L Insulin, asecond feed at day 7 containing 10.8 L of 2× basal media (e.g. CD CHOmedia), 33 g/L Glucose, 16 mM L-alanyl-L-glutamine, 33.4 g/L Yeastextract and 0.92 g/L Sodium Butyrate, a third feed at day 9 containing10.8 L 1× basal media (e.g. CD CHO media), 50 g/L Glucose, 10 mML-alanyl-L-glutamine, 50 g/L Yeast extract and 1.80 g/L Sodium Butyrate,and a fourth feed at day 11 containing 1× basal media (e.g. CD CHOmedia), 33 g/L Glucose, 6.6 mM L-alanyl-L-glutamine, 50 g/L Yeastextract and 0.92 g/L Sodium Butyrate. This can be scaled up or down byone of skill in the art for production of rHuPH20 in larger or smallerbioreactors, respectively. Further, one of skill in the art can alterthe amount of type of one more supplements added to the media to enhancecell growth and/or protein production.

In another example, the following feed media are provided to cells ondays 5, 7, 9 and 11: Feed #1 Medium: basal media+33 g/L Glucose+26.6 mML-alanyl-L-glutamine+83.3 g/L Yeastolate+33 mg/L rHuInsulin; Feed #2:basal media+33 g/L Glucose+13.4 mM L-alanyl-L-glutamine+166.7 g/LYeastolate+0.92 g/L Sodium Butyrate; Feed #3: basal media+50 g/LGlucose+10 mM L-alanyl-L-glutamine+250 g/L Yeastolate+1.8 g/L SodiumButyrate; Feed #4: basal media+33.3 g/L Glucose+6.7 mML-alanyl-L-glutamine+250 g/L Yeastolate+0.92 g/L Sodium butyrate.

G. Cell Culture Harvest, Protein Concentration and Buffer Exchange

Following the protein production phase, the cells are harvested and thesoluble rHuPH20 that has been secreted into the cell culture media isconcentrated prior to initiation of the purification process. Inaddition to concentrating the protein, the cell culture media can beexchanged with an appropriate buffer at this time. Multiple systems andprocesses to effect protein concentration and buffer exchange are knownin the art and can be used in the methods herein. Described below areexemplary methods of such, and one of skill in the art will recognizethat these methods can be modified or substituted with other effectivemethods to achieve a satisfactory level of protein concentration andbuffer exchange.

The cells are harvested from the bioreactor and processed through a cellremoval and clarification system to separate the cell culture fluidcontaining the hyaluronidase from cells and cell debris. An example ofsuch a system is one that contains a series of filters that allow onlythe protein to pass though and be collected. Any filter or series offilters capable of separating the hyaluronidase from cells and celldebris can be used. For example, the cell culture harvest can be passedthrough a series of capsule filters, such a polyethersulfone filters.These can have decreasing pore sizes to incrementally remove, forexample, cells, cell debris and smaller particles, such as viruses. Insome examples, a series of four filters with pore sizes of 8.0 μm, 0.65μm, 0.22 μm and 0.22 μm is used to clarify the cell culture to obtainthe harvested cell culture fluid (HCCF). Another example of a cellremoval and clarification system that can be used in the methods hereinis a series of filters that in the first stage contains four modules inparallel, each containing a layer of diatomaceous earth graded to 4-8 μmand a layer of diatomaceous earth graded to 1.4-1.1 μm, followed by acellulose membrane. The second stage contains a single module containinga layer of diatomaceous earth graded to 0.1-0.11 μm and a layer ofdiatomaceous earth graded to <0.1 μm followed by a cellulose membrane,and the third stage is a 0.22 μm polyethersulfone capsule filter.

Once the cells and debris have been separated from the HCCF, the proteinin the HCCF typically is concentrated and the cell culture mediaexchanged with an appropriate buffer. The protein can be concentrated by2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13× or more. In someexamples, the protein is concentrated 10×. In other examples, theprotein is concentrated 6×. Any method of protein concentration known inthe art can be utilized. Exemplary of such methods include concentrationusing tangential flow filtration (TFF) systems with molecular weight cutoff (MWCO) filters. For example, the clarified HCCF can be passedthrough a series of two 30 kDa MWCO spiral polyethersulfone filters toconcentrate the protein 10×. In another example, the HCCF is passedthrough a series of four 30 kDa MWCO filters. For large-scale productionof hyaluronidase, such as, for example, 100 L and 300 L cultures,filters with surface areas of between 0.5 and 5 square meters aretypically employed for this purpose. In some examples, filters with asurface area of 1.2 square meters or 2.8 square meters are used.

A buffer exchange is performed following protein concentration. One ofskill in the art can empirically determine an appropriate buffer.Exemplary of suitable buffers is a 10 mM Hepes, 25 mM NaCl, pH 7.0buffer, or a 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 buffer. Followingharvesting, concentration and buffer exchange, the concentrated proteinsolution is typically passed through another filter, such as a 0.22 μmcapsule filter, before being stored in a sterile storage bag.

In some examples, the concentrated protein solution is treated toinactivate any residual virus contamination. Virus inactivation can beeffected by any method known in the art. For example, the concentratedprotein solution can be mixed with a 10% Triton X-100, 3% tri (n-butyl)phosphate (TNBP), to a final concentration of 1% Triton X-100, 0.3%TNBP, at room temperate for between 15 and 75 minutes. In some examples,the protein is exposed to the viral inactivation solution for 30-45minutes.

H. Purification

The soluble rHuPH20 is purified from the concentrated protein solutionusing a series of purification steps. Many purification techniques areknown in the art and can be utilized in the methods herein. Such methodscan include, but are not limited to, chromatographic methods such asion-exchange chromatography, size-exclusion chromatography, affinitychromatography (AC), high performance liquid chromatography (HPLC),reversed phase chromatography (RPC) and hydrophobic interactionchromatography (HIC), and gel filtration methods, or any combinationthereof.

Exemplary of purification methods that are used for the methods hereinis a combination of ion-exchange chromatography, hydrophobic interactionchromatography and affinity chromatography. In ion-exchangechromatography, the proteins can be separated from a complex solution ormixture based on electrostatic forces between charged functional groupsof the proteins and charged functional groups of thechromatography-column matrix. Cation-exchange resins have negativelycharged functional groups that attract positively charged functionalgroups of proteins, and anion-exchange resins have positively chargedfunctional groups that attract negatively charged functional groups ofproteins. Proteins bound through electrostatic forces to the matrix canbe eluted by increasing the ionic strength of the buffer solution withinthe chromatography column over time. In hydrophobic interactionchromatography, a protein can be separated from a complex solution ormixture based on its hydrophobicity. A complex solution containing theprotein is applied to a chromatography column equilibrated with a highsalt buffer that facilitates binding of the protein to the resin. Asalt-gradient mobile phase with decreasing ionic strength is thenintroduced into the chromatography column to release bound proteins fromthe matrix. Alternatively, hydrophobic interaction chromatography mayseparate a monomeric protein from a complex solution or mixture bybinding hydrophobic impurities, including inactive dimers and aggregatesof the protein, while permitting the monomeric protein to flow throughthe chromatography column relatively unimpeded. In affinitychromatography, a proteins can be separated from a complex solutionbased on the affinity of the protein for a ligand or ligand-bindingentity that is covalently bound to the matrix. Other proteins in thecomplex solution or mixture with weak affinity, or lacking affinity, forthe ligand or ligand-binding entity flow through the chromatographycolumn unimpeded, leaving the protein of interest bound to the matrix.The protein can then be eluted from the chromatography column byaltering buffer conditions to decrease the affinity for the ligand orligand-binding entity.

In one example, the soluble rHuPH20 is purified from the concentratedprotein solution by sequential purification through a beaded crosslinkedagarose column, such as a Q Sepharose™ column (ion-exchangechromatography), beaded crosslinked phenyl-substituted agarose column,such as a Phenyl Sepharose™ column (hydrophobic interactionchromatography), an Amino Phenyl Boronate column (affinitychromatography) and finally through a Hydroxyapatite column(ion-exchange chromatography). Each of these columns exhibit differentbinding properties with regards to hyaluronidase, such that the beadedcrosslinked agarose column (e.g. Q Sepharose™ column) is a capture step(i.e. soluble rHuPH20 is bound to the resin while some other proteinsflow through), the beaded crosslinked phenyl-substituted agarose (e.g.Phenyl Sepharose™ column) is a flow through step (i.e. soluble rHuPH20flows through the column while some other proteins are captured), theAmino Phenyl Boronate column is another capture step, and theHydroxyapatite column is a polishing step to further purify the solublerHuPH20.

Prior to use, the columns are typically sterilized and the equilibrated.Sterilization can be effected by any method known in the art, including,but not limited to, sterilization with 1.0 M NaOH. Equilibration can beeffected by the addition of an appropriate buffer to the column, such asa buffer similar to or the same as the buffer used to subsequently washthe column or the buffer in which the protein is contained in prior toloading. One of skill in the art can readily determine buffers suitablefor use in equilibrating each column. Exemplary buffers are providedbelow. Between each chromatography step, the eluted protein can befiltered, such as through a 0.22 μm filter, to remove any contaminatingmicroorganism or large aggregates. In some examples, the filtered eluateis stored, such as in sterile storage bags, prior to use in the nextstep. Following column chromatography, the purified hyaluronidase cansubsequently be subjected to a virus removal step, followed by proteinconcentration and buffer exchange for final formulation. Exemplarypurification methods are described in more detail below.

1. Beaded Crosslinked Agarose Column

The concentrated protein obtained from the of harvested cell culturefluid (HCCF) can be loaded onto a beaded crosslinked agarose column,such as, for example, a Q Sepharose™ column, which is a strong anionexchanger and captures soluble rHuPH20 while allowing other proteins toflow through. The bound soluble rHuPH20 can then be eluted using anappropriate buffer. The dimensions of the column used is typicallydependent on the volume of concentrated protein obtained from the HCCF.For example, concentrated protein obtained from culture ofhyaluronidase-expressing cells in a 100 L bioreactor culture can beloaded onto a column that is 20 cm high, 14 cm in diameter and contains3 L resin. In another example, concentrated protein obtained fromculture of soluble rHuPH20-expressing cells in a 300 L bioreactorculture can be loaded onto a column that is 29 cm high, 20 cm indiameter and contains 9 L resin. This can be scaled up or down asnecessary, depending on the volume of the concentrated protein solutionand the expected amount of protein. For example, concentrated proteinobtained from culture of soluble rHuPH20-expressing cells in a 20 Lbioreactor culture can be loaded onto a Q Sepharose™ column that is 28cm high, 7 cm in diameter and contains 1.1 L resin, and concentratedprotein obtained from culture of soluble rHuPH20-expressing cells in a2500 Lbioreactor culture can be loaded onto a Q Sepharose™ column thatis 26 cm high, 63 cm in diameter and contains 81 L resin

Prior to loading with the protein, the column is typically equilibrated.Equilibration can be effected by passing through 1, 2, 3, 4, 5, 6, 7, 8,9 or more column volumes of buffer. In some examples, 5 column volumesof buffer is passed through the column for equilibration. Bufferssuitable for equilibration include those similar to the buffers thatwill be used to wash the column after the protein had been loaded. Forexample, a beaded crosslinked agarose column, such as a Q Sepharose™column can be equilibrated with 10 mM Hepes, 25 mM NaCl, pH 7.5. Otherneutral pH buffers can be used, as will be recognized by one of skill inthe art.

After loading the protein concentrate, the column is washed and theprotein eluted. Suitable buffers for washing such columns containingbound soluble rHuPH20 include, for example, 10 mM Hepes, 25 mL NaCl, pH7.0; 10 mM Hepes, 50 mM NaCl, pH 7.0; and 10 mM Tris, 20 mM Na₂SO₄, pH7.5. The column can be washed with one or more types of buffer. Forexample, the column can be washed with 20 mM Na₂SO₄, pH 7.5 and 10 mMHepes, 50 mM NaCl, pH 7.0. Typically, washing is effected by passingthrough 1, 2, 3, 4, 5, 6, 7, 8, 9 or more column volumes of buffer. Insome examples, 5 column volumes of buffer is used to wash the column.The soluble rHuPH20 is then eluted using a buffer with a higher saltconcentration, such as for example, 10 mM Hepes, 400 mM NaCl, pH 7.0. Insome examples, the absorbance at A₂₈₀ is monitored to determine when tocollect the eluate, as any absorbance during this process generallyindicates the presence of soluble rHuPH20. Thus, in one example, theeluate is collected when the absorbance begins reading is 0.025.Typically, the eluate is filtered through an appropriate filter, such asa 0.22 μm filter, before being stored, such as in a sterile storage bag.

2. Beaded Crosslinked Phenyl-substituted Agarose Column

Following purification through a beaded crosslinked agarose column, theprotein solution can be subjected to hydrophobic interactionchromatography using a beaded crosslinked phenyl-substituted agarosecolumn, such as a Phenyl Sepharose™ column, in which the soluble rHuPH20flows through the column while other contaminating proteins arecaptured. The column used in the methods herein can range in size,depending on the volume and amount of protein being purified though it.Exemplary sizes include columns that are 29 cm high, 20 cm in diameterwith 9 L resin for use in the purification of soluble rHuPH20 from cellsgrown in a 100 L bioreactor culture, columns that are 29 cm high, 30 cmin diameter with 19-21 L resin for use in the purification ofhyaluronidase from cells grown in a 300 L bioreactor culture, andcolumns that are 35 cm high, 80 cm in diameter with 176 L resin for usein the purification of soluble rHuPH20 from cells grown in a 2500 Lbioreactor culture. One of skill in the art can scale up or down asappropriate.

The sterilized beaded crosslinked phenyl-substituted agarose column,such as a Phenyl Sepharose™ column, can be equilibrated prior to loadingof the protein with an appropriate buffer, such as, for example, 5 mMpotassium phosphate, 0.5 M ammonium sulfate, 0.1 mM CaCl₂, pH 7.0. Theprotein eluate from the Q Sepharose column purification also issupplemented with ammonium sulfate, potassium phosphate and CaCl₂. Thesecan be supplemented to the protein to final concentrations of, forexample, about 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1mM CaCl₂, pH 7.0. Following loading of the protein, 5 mM potassiumphosphate, 0.5 M ammonium sulfate and 0.1 mM CaCl₂, pH 7.0 also is addedto the column and the flow through filtered is collected, such as in asterile bag.

3. Amino Phenyl Boronate Column

Following hydrophobic interaction chromatography, the column-purifiedprotein can be loaded onto an Amino Phenyl Boronate column for furtherpurification. Amino Phenyl boronate ligand-mediated chromatographydiffers from many other ligands used for affinity chromatography.Whereas most ligands bind to a particular binding site on a protein by amixture of noncovalent interactions, phenyl boronate interactspredominantly by forming a temporary covalent bond with 1,2-cis-diolgroups. The boronate ligand will bind to any molecule containing theappropriate group, including soluble rHuPH20, which is highlyglycosylated.

The Amino Phenyl Boronate column used in the methods herein can range insize, depending on the volume and amount of protein being purifiedthough it. Exemplary sizes include columns that are 29 cm high, 20 cm indiameter with 6.3 L resin for use in the purification of hyaluronidasefrom cells grown in a 100 L bioreactor culture, columns that are 29 cmhigh, 30 cm in diameter with 19-21 L resin for use in the purificationof hyaluronidase from cells grown in a 300 L bioreactor culture, and,and columns that are 35 cm high, 80 cm in diameter with 176 L resin foruse in the purification of hyaluronidase from cells grown in a 2500 Lbioreactor culture. One of skill in the art can scale up or down asappropriate. Buffers suitable for equilibrating the Amino PhenylBoronate column include, for example, buffers containing 5 mM potassiumphosphate, 0.5 M ammonium sulfate, pH 7.0.

Following loading of the Phenyl Sepharose column-purified protein ontothe Amino Phenyl Boronate column, the column is washed with suitablewash buffers. Exemplary wash buffers include, but are not limited to, 5mM potassium phosphate, 0.5 M ammonium sulfate, pH 7.0, and 20 mMbicine, 0.5 M ammonium sulfate, pH 9.0 and 20 mM bicine, 100 mM NaCl, pH9.0. In one example, the Amino Phenyl Boronate column with the boundhyaluronidase is washed first with 5 mM potassium phosphate, 0.5 Mammonium sulfate, pH 7.0, then with 20 mM bicine, 0.5 M ammoniumsulfate, pH 9.0 and finally with 20 mM bicine, 100 mM NaCl, pH 9.0. Thebound hyaluronidase can then be eluted, such as with 50 mM Hepes, 100 mMNaCl, pH 7.0. One of skill in the art can modify one or more of thebuffers to similarly effect purification. Typically, the eluted solublerHuPH20 also is filtered to remove any microbial contamination or largeaggregates.

4. Hydroxyapatite Column

Following Phenyl Boronate column chromatography, the protein solutioncontaining the soluble rHuPH20 can be loaded onto a Hydroxyapatitecolumn in a final polishing step. Hydroxyapatite is a crystalline formof calcium phosphate with the molecular formula Ca₁₀(PO₄)₆(OH)₂. It canbe used as a polishing step to separate closely copurifying proteins,operating by mixed-mode ion exchange due to its inclusion of bothpositively and negatively charged moieties. Various hydroxyapatitechromatographic media are available commercially, and any available formof the material can be used in the methods herein. Examples ofhydroxyapatites include, but are not limited to, those that areagglomerated to form particles and sintered at high temperatures into astable porous ceramic mass. The particle size can vary, such as rangingfrom about 1 μm to about 1,000 μm in diameter. The porosity also canalso vary, such as from about 100 A to about 10,000 A.

The Hydroxyapatite column used in the methods herein can range in size,depending on the volume and amount of protein being purified though it.Exemplary sizes include columns that are 20 cm high, 30 cm in diameterwith 13 L resin for use in the purification of hyaluronidase from cellsgrown in a 300 L bioreactor culture, and columns that are 23 cm high, 80cm in diameter with 116 L resin for use in the purification ofhyaluronidase from cells grown in a 2500 L bioreactor culture. One ofskill in the art can scale up or down as appropriate.

For the methods described herein, the Hydroxyapatite column can beequilibrated with 5 mM potassium phosphate, 200 mM NaCl or 5 mMpotassium phosphate, 200 mM NaCl, 0.1 mM CaCl₂, pH 7.0. Equilibrationusing solutions such as these make the column compatible with thepartially-purified hyaluronidase, which itself is supplemented withpotassium phosphate and CaCl₂ to final concentrations of 5 mM and 0.1mM, respectively. Following loading of the protein onto the column, thecolumn can be washed with, for example, 10 mM potassium phosphate, 100mM NaCl, 0.1 mM CaCl₂, pH 7.0, to remove any unbound contaminatingproteins. The bound soluble rHuPH20 can then be eluted with anappropriate elution buffer. For example, elution can be effected by theaddition of 70 mM potassium phosphate, pH 7.0. In some examples, theeluate is filtered, such as through a 0.22 μm filter.

6. Virus Removal, Protein Concentration and Buffer Exchange

The soluble rHuPH20 obtained flowing column chromatography can besubjected to post-purification steps that serve to formulate the proteinin the desired buffer at the desired concentration. The protein also canbe subjected to a viral removal step to ensure it is free fromcontamination and suitable for use as a therapeutic. Viral removal istypically effected with the use of a filter that allows only the solubleprotein to pass through while trapping any viruses (and othercontaminants that are equal to in size or larger that viruses). Suchfilters are available commercially, and any can be used in the methodsherein. Pores sizes of filters useful for viral removal include, but arenot limited to, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm,75 nm and 100 nm. In one example, the purified hyaluronidase is filteredthrough a filter containing 20 nm pores. The protein can be pumped intothe filter by, for example, peristaltic pump or by use of a pressuretank.

Following viral removal, the soluble rHuPH20 can be concentrated andsubjected to buffer exchange. The soluble rHuPH20 can be concentrated by2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13× or more. In someexamples, the protein is concentrated approximately 6×. This can resultin, for example, a concentration of between 0.1 mg/mL and 50 mg/mL. Insome examples, the purified hyaluronidase is concentrated toapproximately 1 mg/mL. In other examples, the purified hyaluronidase isconcentrated to approximately 10 mg/mL. Any method of proteinconcentration known in the art can be utilized. Exemplary of suchmethods include concentration using tangential flow filtration (TFF)systems with molecular weight cut off (MWCO) filters. For example, thepurified hyaluronidase can be passed through a 10 kDa MWCO spiralpolyethersulfone filters to concentrate the protein 10×. In anotherexample, the protein is passed through a series of four 30 kDa MWCOfilters. For large-scale production of hyaluronidase, such as, forexample, 100 L and 300 L cultures, filters with surface areas of between0.5 and 5 square meters are typically employed for this purpose. In someexamples, filters with a surface area of 1.2 square meters or 2.8 squaremeters are used.

A buffer exchange is generally performed following protein concentrationto formulate the protein in the desired buffer for subsequent use, forexample, as a therapeutic. One of skill in the art can empiricallydetermine an appropriate buffer. Exemplary of suitable buffers aresaline buffers, including, but not limited to, 10 mM Hepes, 130 mM NaCl,pH 7.0, and 10 mM Histidine, 130 mM NaCl, pH 6.0. The purifiedhyaluronidase can, in some example, be passed through another filter,such as a 0.22 μm capsule filter, before being stored in a sterileenvironment.

I. Filling

The methods described herein for the production and purification ofsoluble rHuPH20 also can include a filling step, in which the purifiedprotein is aseptically filled into smaller containers for long-termstorage and use. The soluble rHuPH20 can be filled into the containersas a liquid formulation, or as a powder, such as followinglyophilization. For large-scale production, automated filling systemsthat include, for example, pumps to transfer the protein to thecontainers and weighing stations to measure the fill volume aretypically used and are widely available. Manual or a combination ofautomated and manual filling of containers also can be performed,however. Suitable containers include, but are not limited to, glass orplastic vials, blister packs, bottles, tubes, inhalers, pumps, bags,syringes, bottles, or any other suitable container. Suitable closures orcaps also can be used to seal the container. The filling process caninclude first passing the soluble rHuPH20 through a filter prior tofilling to remove microbial contaminant and larger aggregates orsediment. For example, the protein can be filtered through a 0.22 μmfilter before being aliquoted into suitable containers. One of skill inthe art can determine the appropriate fill volume and can include, forexample, volumes ranging from 0.1 mL to 100 mL. In some examples, vialsare asceptically filled with 1 mL, 5 mL or 20 mL soluble rHuPH20.Following capping or closure of the containers, the containers can bestored at an appropriate temperature. In some examples, the containersare flash frozen and stored at between −15° C. and −35° C. In otherexamples, the containers are refrigerated, such as at between 3° C. and15° C. Typically, long-term storage of liquids is at lower temperaturesto minimize degradation. Soluble rHuPH20 in powder form can be storedfor long periods at room temperature without significant degradation.

J. Monitoring and Assays

The methods described herein can be monitored at one or more steps,measuring one or more conditions, parameters or products at each point.This can ensure that optimal conditions are maintained throughout, andalso can be used to assess efficiency and productivity of the process.Monitoring can occur, for example, one or more times during the cellexpansion phase, protein production phase (i.e. in the bioreactor),and/or the protein purification stage, as well as any time between,before or after, such as during concentration/buffer exchange proceduresor filling. Monitoring can include, but is not limited to, measuring pH,temperature, volumes, contamination, purity, protein concentration,enzyme activity, cell viability and cell number. In addition tomonitoring conditions, parameters or products throughout the process,the purified soluble rHuPH20 produced as the end product also can beassessed and characterized with respect to, for example, proteinconcentration, enzyme activity, impurities, contamination, osmolarity,degradation, post-translational modifications and monosaccharidecontent.

1. Monitoring the Conditions

The conditions during one or more of the steps in the methods providedherein can be monitored to ensure optimal conditions are maintainedthroughout the process. If the monitoring demonstrates that theconditions are not within an optimal range, then the conditions can bealtered. Conditions that can be monitored vary for each process. Forexample, during the cell culture phases (i.e. cell expansion and proteinproduction in the bioreactor), conditions to be monitored include, butare not limited to, temperature, cell culture pH, cell culture nutrients(e.g. glucose), CO₂ levels and O₂ levels. Typically, the conditions aremonitored automatically using in-built systems in, for example theincubator or bioreactor.

During the protein purification stage, conditions that can be monitoredinclude, but are not limited to, pH, conductivity and flow rate. Theseconditions can be monitored before, during and/or after one or morecolumn chromatography steps. For example, the buffers used toequilibrate, wash or elute the column can be monitored. This can beperformed before the buffer is loaded or after the buffer has runthrough the column.

2. Monitoring Soluble rHuPH20 Production

Soluble rHuPH20 production, and parameters associated with solublerHuPH20 production, also can be monitored throughout the process. Theseinclude, but are not limited to, cell number, cell viability,contamination, protein concentration, enzyme activity, purity,osmolarity, post-translational modifications. Any method to assess theseparameters can be used. For example, mammalian cell viability can beassessed by taking a small aliquot of the cell culture and staining withtrypan blue, which permeates only damaged cell membranes, thus stainingonly dead cells. The cells can be visualized under microscope andcounted using, for example, a hemocytometer. Other methods includeassessing cell viability by measuring metabolic activity. For example,an aliquot of the cell culture can be incubated with a tetrazolium salt(e.g. MTT, XTT or WST-1) that is cleaved into a colored formazan productby metabolically active cells.

Soluble rHuPH20 concentration in a particular sample can be assessed bymethods well-known in the art, including but not limited to,enzyme-linked immunosorbant assays (ELISA); SDS-PAGE; Bradford, Lowry,and/or BCA methods; UV absorbance, and other quantifiable proteinlabeling methods, such as, but not limited to, immunological,radioactive and fluorescent methods and related methods. Additionally,the presence and extent of degradation can be measured by standardtechniques such as sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), Western blotting of electrophoresedhyaluronidase-containing samples and chromatography, such as, forexample, RP-HPLC. The purity of a hyaluronidase-containing sample can beassessed by, for example, SDS-PAGE, RP-HPLC, size-exclusionchromatography, anion-exchange chromatography and isoelectric focusing(IEF). Soluble rHuPH20-containing samples, such as samples containingpurified hyaluronidase, can be further characterized by assessing thesialic acid and monosaccharide content. This can be accomplished by, forexample, hydrolyzing the sample with 40% trifluoroacetic acid,fluorescently labeling the released monosaccharides and separating themusing RP-HPLC (see Example 10).

Soluble rHuPH20 produced and purified using the methods provided hereinalso can be assessed for the presence of post-translationalmodifications. Such assays are known in the art and include assays tomeasure glycosylation, hydroxylation, and carboxylation. In an exemplaryassay for glycosylation, carbohydrate analysis can be performed, forexample, with SDS page analysis of soluble rHuPH20 exposed tohydrazinolysis or endoglycosidase treatment. Hydrazinolysis releases N-and O-linked glycans from glycoproteins by incubation with anhydroushydrazine, while endoglycosidase release involves PNGase F, whichreleases most N-glycans from glycoproteins. Hydrazinolysis orendoglycosidase treatment of soluble rHuPH20 polypeptides generates areducing terminus that can be tagged with a fluorophore or chromophorelabel. Labeled soluble rHuPH20 polypeptides can be analyzed byfluorophore-assisted carbohydrate electrophoresis (FACE). Thefluorescent tag for glycans also can be used for monosaccharideanalysis, profiling or fingerprinting of complex glycosylation patternsby HPLC. Exemplary HPLC methods include hydrophilic interactionchromatography, electronic interaction, ion-exchange, hydrophobicinteraction, and size-exclusion chromatography. Exemplary glycan probesinclude, but are not limited to, 3-(acetylamino)-6-aminoacridine (AA-Ac)and 2-aminobenzoic acid (2-AA). Carbohydrate moieties can also bedetected through use of specific antibodies that recognize theglycosylated hyaluronidase polypeptide.

An exemplary assay to measure β-hydroxylation comprises reverse phaseHPLC analysis of soluble rHuPH20 polypeptides that have been subjectedto alkaline hydrolysis (Przysiecki et al. (1987) PNAS 84:7856-7860).Carboxylation and y-carboxylation of hyaluronidase polypeptides can beassessed using mass spectrometry with matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) analysis, as described in the art(se, e.g. Harvey et al. J Biol Chem 278:8363-8369, Maum et al. Prot Sci14:1171-1180).

The enzymatic activity of soluble rHuPH20 in a sample can be assessed atany point during the methods described herein. In one example, activityis measured using a microturbidity assay (see e.g. Example 10). This isbased on the formation of an insoluble precipitate when hyaluronic acidbinds with serum albumin. The activity is measured by incubating solublerHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period oftime (e.g. 10 minutes) and then precipitating the undigested sodiumhyaluronate with the addition of acidified serum albumin. The turbidityof the resulting sample is measured at 640 nm after an additionaldevelopment period. The decrease in turbidity resulting from enzymeactivity on the sodium hyaluronate substrate is a measure of the solublerHuPH20 enzymatic activity. In another example, enzymatic activity ismeasured using a microtiter assay in which residual biotinylatedhyaluronic acid is measured following incubation with the samplecontaining soluble rHuPH20 (see e.g. Frost and Stern (1997) Anal.Biochem. 251:263-269, U.S. Patent Publication No. 20050260186). The freecarboxyl groups on the glucuronic acid residues of hyaluronic acid arebiotinylated, and the biotinylated hyaluronic acid substrate iscovalently couple to a microtiter plate. Following incubation with thesample containing soluble rHuPH20, the residual biotinylated hyaluronicacid substrate is detected using an avidin-peroxidase reaction, andcompared to that obtained following reaction with hyaluronidasestandards of known activity. Other assays to measure enzymatic activityalso are known in the art and can be used in the methods herein (seee.g. Delpech et al., (1995) Anal. Biochem. 229:35-41; Takahashi et al.,(2003) Anal. Biochem. 322:257-263).

The presence of any contamination also can monitored. Contamination caninclude, but is not limited to, microbial contamination (e.g. viruses,bacteria and mycoplasma), microbial products contamination (e.g.endotoxin), or other process-related impurities. Any suitable method orassay can be used. For example, viruses and bacteria can be culturedusing methods well known in the art to determine whether they arepresent or not in a sample, and if so, in what quantities. Microscopyalso can be used to detect microbial contamination. For example, asample can be assessed for the presence of viruses or bacteria usingtransmission electron microscopy (TEM). Mycoplasma detection can beeffected using, for example, biochemical or molecular techniques,including, but not limited to, PCR to amplify mycoplasma-specificnucleic acid, biochemical tests to detect mycoplasmal enzymes andcell-based fluorescence to detect mycoplasmal antigens or nucleic acid.

The presence of microbial products, such as bacterial endotoxins, alsocan be monitored. An example of a suitable assay for detecting thepresence of endotoxin is the Limulus Amebocyte Lysate (LAL) assay. Twotypes of LAL assays can be used: gel clot and photometric (chromogenicand turbometric). LAL is an aqueous extract of blood cells (amebocytes)from the horse shoe crab. Endotoxin triggers a cascade of enzymaticreactions, which result in activated clotting enzyme. In the presence ofbacterial endotoxins, at an elevated temperature, the LAL reagent willclot after addition of reagent. The formation of the gel clot isproportional to the concentration of the endotoxin. In the kineticassay, the proenzyme in the LAL is activated when in contact withendotoxins produced by gram negative bacteria. The rate of activation isdirectly proportional with the concentration of the endotoxin present.The level of activation can be measured through a subsequent substratereaction which is measured spectrophotometrically.

K. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Generation of a Soluble rHuPH20-Expressing Cell Line

The HZ24 plasmid (set forth in SEQ ID NO:50) was used to transfectChinese Hamster Ovary (CHO cells) (see e.g. related application Nos.10,795,095, 11/065,716 and 11/238,171). The HZ24 plasmid vector forexpression of soluble rHuPH20 contains a pCI vector backbone (Promega),DNA encoding amino acids 1-482 of human PH20 hyaluronidase (SEQ IDNO:47), an internal ribosomal entry site (IRES) from the ECMV virus(Clontech), and the mouse dihydrofolate reductase (DHFR) gene. The pCIvector backbone also includes DNA encoding the Beta-lactamase resistancegene (AmpR), an f1 origin of replication, a Cytomegalovirusimmediate-early enhancer/promoter region (CMV), a chimeric intron, andan SV40 late polyadenylation signal (SV40). The DNA encoding the solublerHuPH20 construct contains an NheI site and a Kozak consensus sequenceprior to the DNA encoding the methionine at amino acid position 1 of thenative 35 amino acid signal sequence of human PH20, and a stop codonfollowing the DNA encoding the tyrosine corresponding to amino acidposition 482 of the human PH20 hyaluronidase set forth in SEQ ID NO:1),followed by a BamHI restriction site. The constructpC1-PH20-IRES-DHFR-SV40pa (HZ24), therefore, results in a single mRNAspecies driven by the CMV promoter that encodes amino acids 1-482 ofhuman PH20 (set forth in SEQ ID NO:3) and amino acids 1-186 of mousedihydrofolate reductase (set forth in SEQ ID NO:51), separated by theinternal ribosomal entry site (IRES).

Non-transfected DG44 CHO cells growing in GIBCO Modified CD-CHO mediafor DHFR(−) cells, supplemented with 4 mM Glutamine and 18 ml/LPlurionic F68/L (Gibco), were seeded at 0.5×10⁶ cells/ml in a shakerflask in preparation for transfection. Cells were grown at 37° C. in 5%CO₂ in a humidified incubator, shaking at 120 rpm. Exponentially growingnon-transfected DG44 CHO cells were tested for viability prior totransfection.

Sixty million viable cells of the non-transfected DG44 CHO cell culturewere pelleted and resuspended to a density of 2×10⁷ cells in 0.7 mL of2× transfection buffer (2×HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mMKCl, 1.4 mM Na₂HPO₄, 12 mM dextrose). To each aliquot of resuspendedcells, 0.09 mL (250 μg) of the linear HZ24 plasmid (linearized byovernight digestion with Cla I (New England Biolabs) was added, and thecell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics)electroporation cuvettes at room temperature. A negative controlelectroporation was performed with no plasmid DNA mixed with the cells.The cell/plasmid mixes were electroporated with a capacitor discharge of330 V and 960 μF or at 350 V and 960 μF.

The cells were removed from the cuvettes after electroporation andtransferred into 5 mL of Modified CD-CHO media for DHFR(−) cells,supplemented with 4 mM Glutamine and 18 ml/L Plurionic F68/L (Gibco),and allowed to grow in a well of a E-well tissue culture plate withoutselection for 2 days at 37° C. in 5% CO₂ in a humidified incubator.

Two days post-electroporation, 0.5 mL of tissue culture media wasremoved from each well and tested for the presence of hyaluronidaseactivity, using the microturbidity assay described in Example 9.

TABLE 1 Initial Hyaluronidase Activity of HZ24 Transfected DG44 CHOcells at 40 hours post-transfection Activity Dilution Units/mlTransfection 1 1 to 10 0.25 330 V Transfection 2 1 to 10 0.52 350 VNegative 1 to 10 0.015 Control

Cells from Transfection 2 (350V) were collected from the tissue culturewell, counted and diluted to 1×10⁴ to 2×10⁴ viable cells per mL. A 0.1mL aliquot of the cell suspension was transferred to each well of five,96 well round bottom tissue culture plates. One hundred microliters ofCD-CHO media (GIBCO) containing 4 mM GlutaMAX™-I supplement (GIBCO™,Invitrogen Corporation) and without hypoxanthine and thymidinesupplements were added to the wells containing cells (final volume 0.2mL).

Ten clones were identified from the 5 plates grown without methotrexate.

TABLE 2 Hyaluronidase activity of identified clones Relative Plate/WellID Hyaluronidase 1C3 261 2C2 261 3D3 261 3E5 243 3C6 174 2G8 103 1B9 3042D9 273 4D10 302

Six HZ24 clones were expanded in culture and transferred into shakerflasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1E11, and4D10 were plated into 96-well round bottom tissue culture plates using atwo-dimensional infinite dilution strategy in which cells were diluted1:2 down the plate, and 1:3 across the plate, starting at 5000 cells inthe top left hand well. Diluted clones were grown in a background of 500non-transfected DG44 CHO cells per well, to provide necessary growthfactors for the initial days in culture. Ten plates were made persubclone, with 5 plates containing 50 nM methotrexate and 5 plateswithout methotrexate.

Clone 3D3 produced 24 visual subclones (13 from the no methotrexatetreatment, and 11 from the 50 nM methotrexate treatment. Significanthyaluronidase activity was measured in the supernatants from 8 of the 24subclones (>50 Units/mL), and these 8 subclones were expanded into T-25tissue culture flasks. Clones isolated from the methotrexate treatmentprotocol were expanded in the presence of 50 nM methotrexate. Clone3D35M was further expanded in 500 nM methotrexate giving rise to clonesproducing in excess of 1,000 Units/ml in shaker flasks (clone 3D35M; orGen1 3D35M). A master cell bank (MCB) of the 3D35M cells was thenprepared.

Example 2 Determination of the Copy Number of the Nucleic Acid RegionEncoding Soluble rHuPH20 in 3D35M Cells

The copy number of the nucleic acid region encoding soluble rHuPH20 in3D35M cells was determined by quantitative PCR. Total genomic DNA wasextracted from 3D35M cells from the MCB. Six independent dilutions ofthe DNA were prepared for analysis in duplicate, each of which containedapproximately 6.6 ng DNA (equivalent to approximately 100 cells).Negative controls containing no template also were prepared, as werepositive controls containing the plasmid and the DNA equivalent of 1000CHO cells (6.6 ng). The reactions were assembled according to the TaqManUniversal PCR Master Mix Protocol (Applied Biosystems) and run induplicate. A standard curve was generated using eight dilutions of theHZ24 plasmid, representing a range of approximately 5×10⁶ to 49 copiesof plasmid DNA. The standard was diluted in CHO control genomic DNA(equivalent of 100 cells). The reactions were assembled according to theTaqMan™ Univeral PCR Master Mix Protocol (Applied Biosystems) using theHZM3.P1 forward primer and the HZM3.P2 reverse primer (set forth in SEQID NOS:52 and 53, respectively) and the HZM3 probe (SEQ ID NO:54), whichcontained the fluorescent dyes 6FAM (6-carboxyfluorescin) and TAMRA(6-carboxytetramethylrhodamine). The reactions were run in duplicateusing the following cycling conditions: 50° C. for 2 minutes, 95° C. for10 minutes, followed by 40 cycles of 95° C. for 15 seconds and 60° C.for 1 minute. A standard quantitative PCR reaction to assay GAPDH copiesfor each DNA sample also was performed. Data was collected by the ABIPrism 7700™ Sequence Detection System software version 1.9 (AppliedBiosystems).

The target gene copy numbers per cell were calculated as the ratio oftarget copies (soluble rHuPH20) to normalized copies (GAPDH) for the sixdilutions of 3D35M genomic DNA. The Dixon Q Outlier statistics test wasapplied to the data set. The copy number of the sHuPH20 region in 3D35Mcells was found to be 317.87±11.64.

Example 3 Production and Purification of Gen1 Soluble rHuPH20

A. 5 L Bioreactor Process

A vial of 3D35M was thawed and expanded from T-25 flasks through 1 Lspinner flasks in CD CHO (Invitrogen, Carlsbad Calif.) supplemented with100 nM Methotrexate and 40 mL/L GlutaMAX™-I (Invitrogen; 200 mM stocksolution). Cells were transferred from spinner flasks to a 5 Lbioreactor (Braun) at an inoculation density of 4×10⁵ viable cells perml in 5 L media. Parameters were temperature Setpoint 37° C., pH 7.2(starting Setpoint), with Dissolved Oxygen Setpoint 25% and an airoverlay of 0-100 cc/min. At 168 hrs, 250 ml of Feed #1 Medium (CD CHOwith 50 g/L Glucose) was added. At 216 hours, 250 ml of Feed #2 Medium(CD CHO with 50 g/L Glucose and 10 mM Sodium Butyrate) was added, and at264 hours 250 ml of Feed #2 Medium was added. This process resulted in afinal productivity of 1600 Units per ml with a maximal cell density of6×10⁶ cells/ml. The addition of sodium butyrate was to enhance theproduction of soluble rHuPH20 in the final stages of production.

Conditioned media from the 3D35M clone was clarified by depth filtrationand tangential flow diafiltration into 10 mM Hepes pH 7.0. SolublerHuPH20 was then purified by sequential chromatography on Q Sepharose(Pharmacia) ion exchange, Phenyl Sepharose (Pharmacia) hydrophobicinteraction chromatography, Amino phenyl boronate (ProMetics) andHydroxyapatite Chromatography (Biorad, Richmond, Calif.).

Soluble rHuPH20 bound to Q Sepharose and eluted at 400 mM NaCl in thesame buffer. The eluate was diluted with 2M ammonium sulfate to a finalconcentration of 500 mM ammonium sulfate and passed through a PhenylSepharose (low sub) column, followed by binding under the sameconditions to a phenyl boronate resin. The soluble rHuPH20 was elutedfrom the Phenyl Sepharose resin in Hepes pH 6.9 after washing at pH 9.0in 50 mM bicine without ammonium sulfate. The eluate was loaded onto aceramic hydroxyapatite resin at pH 6.9 in 5 mM potassium phosphate and 1mM CaCl₂ and eluted with 80 mM potassium phosphate, pH 7.4 with 0.1 mMCaCl₂.

The resultant soluble rHuPH20 possessed a specific activity in excess of65,000 Units/mg protein by way of the microturbidity assay (see Example9). Purified soluble rHuPH20 eluted as a single peak from 24 to 26minutes from a Pharmacia 5RPC styrene divinylbenzene column with agradient between 0.1% TFA/H₂O and 0.1% TFA/90% acetonitrile/10% H₂O andresolved as a single broad 61 kDa band by SDS electrophoresis thatreduced to a sharp 51 kDa band upon treatment with PNGASE-F. N-terminalamino acid sequencing revealed that the leader peptide had beenefficiently removed.

B. Upstream Cell Culture Expansion Process into 100 L Bioreactor CellCulture

A scaled-up process was used to separately purify soluble rHuPH20 fromfour different vials of 3D35M cell to produce 4 separate batches ofsHuPH20; HUA0406C, HUA0410C, HUA0415C and HUA0420C. Each vial wasseparately expanded and cultured through a 125 L bioreactor, thenpurified using column chromatography. Samples were taken throughout theprocess to assess such parameters as enzyme yield. The description ofthe process provided below sets forth representative specifications forsuch things as bioreactor starting and feed media volumes, transfer celldensities, and wash and elution volumes. The exact numbers vary slightlywith each batch, and are detailed in Tables 3 to 10.

Four vials of 3D35M cells were thawed in a 37° C. water bath, CD CHOcontaining 100 nM methotrexate and 40 mL/L GlutaMAX™-I was added and thecells were centrifuged. The cells were re-suspended in a 125 mL shakeflask with 20 mL of fresh media and placed in a 37° C., 7% CO₂incubator. The cells were expanded up to 40 mL in the 125 mL shakeflask. When the cell density reached 1.5-2.5×10⁶ cells/mL, the culturewas expanded into a 125 mL spinner flask in a 100 mL culture volume. Theflask was incubated at 37° C., 7% CO₂. When the cell density reached1.5-2.5×10⁶ cells/mL, the culture was expanded into a 250 mL spinnerflask in 200 mL culture volume, and the flask was incubated at 37° C.,7% CO₂. When the cell density reached 1.5-2.5×10⁶ cells/mL, the culturewas expanded into a 1 L spinner flask in 800 mL culture volume andincubated at 37° C., 7% CO₂. When the cell density reached 1.5-2.5×10⁶cells/mL, the culture was expanded into a 6 L spinner flask in 5 Lculture volume and incubated at 37° C., 7% CO₂. When the cell densityreached 1.5-2.5×10⁶ cells/mL, the culture was expanded into a 36 Lspinner flask in 20 L culture volume and incubated at 37° C., 7% CO₂.

A 125 L reactor was sterilized with steam at 121° C., 20 PSI and 65 L ofCD CHO media was added. Before use, the reactor was checked forcontamination. When the cell density in the 36 L spinner flasks reached1.8-2.5×10⁶ cells/mL, 20 L cell culture were transferred from the 36 Lspinner flasks to the 125 L bioreactor (Braun), resulting a final volumeof 85 L and a seeding density of approximately 4×10⁵ cells/mL.Parameters were temperature setpoint, 37° C.; pH: 7.2; Dissolved oxygen:25%±10%; Impeller Speed 50 rpm; Vessel Pressure 3 psi; Air Sparge 1L/min.; Air Overlay: 1 L/min. The reactor was sampled daily for cellcounts, pH verification, media analysis, protein production andretention. Nutrient feeds were added during the run. At Day 6, 3.4 L ofFeed #1 Medium (CD CHO+50 g/L Glucose+40 mL/L GlutaMAX™-I) was added,and culture temperature was changed to 36.5° C. At day 9, 3.5 L of Feed#2 (CD CHO+50 g/L Glucose+40 mL/L GlutaMAX™-I+1.1 g/L Sodium Butyrate)was added, and culture temperature was changed to 36° C. At day 11, 3.7L of Feed #3 (CD CHO+50 g/L Glucose+40 mL/L GlutaMAX™-I+1.1 g/L SodiumButyrate) was added, and the culture temperature was changed to 35.5° C.The reactor was harvested at 14 days or when the viability of the cellsdropped below 50%. The process resulted in production of soluble rHuPH20with an enzymatic activity of 1600 Units/ml with a maximal cell densityof 8 million cells/mL. At harvest, the culture was sampled formycoplasma, bioburden, endotoxin, and virus in vitro and in vivo,transmission electron microscopy (TEM) for viral particles, and enzymeactivity.

The one hundred liter bioreactor cell culture harvest was filteredthrough a series of disposable capsule filters having a polyethersulfonemedium (Sartorius): first through a 8.0 μm depth capsule, a 0.65 μmdepth capsule, a 0.22 μm capsule, and finally through a 0.22 μmSartopore 2000 cm² filter and into a 100 L sterile storage bag. Theculture was concentrated 10× using two TFF with Spiral Polyethersulfone30 kDa MWCO filters (Millipore), followed by a 6× buffer exchange with10 mM HEPES, 25 mM Na₂SO₄, pH 7.0 into a 0.22 μm final filter into a 20L sterile storage bag. Table 3 provides monitoring data related to thecell culture, harvest, concentration and buffer exchange steps.

TABLE 3 Monitoring data for cell culture, harvest, concentration andbuffer exchange steps. Parameter HUA0406C HUA04010C HUA0415C HUA0420CTime from thaw to inoculate 100 L 21 19 17 18 bioreactor (days) 100 Linoculation density (×10⁶ cells/mL) 0.45 0.33 0.44 0.46 Doubling time inlogarithmic 29.8 27.3 29.2 23.5 growth (hr) Max. cell density (×10⁶cells/mL) 5.65 8.70 6.07 9.70 Harvest viability (%) 41 48 41 41 Harvesttiter (U/ml) 1964 1670 991 1319 Time in 100-L bioreactor (days) 13 13 1213 Clarified harvest volume (mL) 81800 93300 91800 89100 Clarifiedharvest enzyme assay 2385 1768 1039 1425 (U/mL) Concentrate enzyme assay22954 17091 8561 17785 (U/mL) Buffer exchanged concentrate 15829 116499915 8679 enzyme assay (U/mL) Filtered buffer exchanged 21550 10882 94718527 concentrate enzyme assay (U/mL) Buffer exchanged concentrate 1069913578 12727 20500 volume(mL) Ratio enzyme units 0.87 0.96 1.32 1.4concentration/harvest

A Q Sepharose (Pharmacia) ion exchange column (3 L resin, Height=20 cm,Diameter=14 cm) was prepared. Wash samples were collected for adetermination of pH, conductivity and endotoxin (LAL) assay. The columnwas equilibrated with 5 column volumes of 10 mM Tris, 20 mM Na₂SO₄, pH7.5. The concentrated, diafiltered harvest was loaded onto the Q columnat a flow rate of 100 cm/hr. The column was washed with 5 column volumesof 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 and 10 mM Hepes, 50 mM NaCl, pH 7.0.The protein was eluted with 10 mM Hepes, 400 mM NaCl, pH 7.0 andfiltered through a 0.22 μm final filter into a sterile bag.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography wasnext performed. A Phenyl-Sepharose (PS) column (9.1 L resin, Height=29cm, Diameter=20 cm) was prepared. The column was equilibrated with 5column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate, 0.1mM CaCl₂, pH 7.0. The protein eluate from above was supplemented with 2Mammonium sulfate, 1 M potassium phosphate and 1 M CaCl₂ stock solutionsto final concentrations of 5 mM, 0.5 M and 0.1 mM, respectively. Theprotein was loaded onto the PS column at a flow rate of 100 cm/hr. 5 mMpotassium phosphate, 0.5 M ammonium sulfate and 0.1 mM CaCl₂ pH 7.0 wasadded at 100 cm/hr. The flow through was passed through a 0.22 μm finalfilter into a sterile bag.

The PS-purified protein was the loaded onto an aminophenyl boronatecolumn (ProMedics) (6.3 L resin, Height=20 cm, Diameter=20 cm) that hadbeen equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5M ammonium sulfate. The protein was passed through the column at a flowrate of 100 cm/hr, and the column was washed with 5 mM potassiumphosphate, 0.5 M ammonium sulfate, pH 7.0. The column was then washedwith 20 mM bicine, 100 mM NaCl, pH 9.0 and the protein eluted with 50 mMHepes, 100 mM NaCl pH 6.9 through a sterile filter and into a 20 Lsterile bag. The eluate was tested for bioburden, protein concentrationand enzyme activity.

A hydroxyapatite (HAP) column (BioRad) (1.6 L resin, Height=10 cm,Diameter=14 cm) was equilibrated with 5 mM potassium phosphate, 100 mMNaCl, 0.1 mM CaCl₂ pH 7.0. Wash samples were collected and tested forpH, conductivity and endotoxin (LAL assay). The aminophenyl boronatepurified protein was supplemented with potassium phosphate and CaCl₂ toyield final concentrations of 5 mM potassium phosphate and 0.1 mM CaCl₂and loaded onto the HAP column at a flow rate of 100 cm/hr. The columnwas washed with 5 mM potassium phosphate pH 7.0, 100 mM NaCl, 0.1 mMCaCl₂, then 10 mM potassium phosphate pH 7.0, 100 mM NaCl, 0.1 mM CaCl₂pH. The protein was eluted with 70 mM potassium phosphate pH 7.0 andfiltered through a 0.22 μm filter into a 5 L sterile storage bag. Theeluate was tested for bioburden, protein concentration and enzymeactivity.

The HAP-purified protein was then pumped through a 20 nM viral removalfilter via a pressure tank. The protein was added to the DV20 pressuretank and filter (Pall Corporation), passing through an Ultipor DV20Filter with 20 nm pores (Pall Corporation) into a sterile 20 L storagebag. The filtrate was tested for protein concentration, enzyme activity,oligosaccharide, monosaccharide and sialic acid profiling, andprocess-related impurities. The protein in the filtrate was thenconcentrated to 1 mg/mL using a 10 kD molecular weight cut off (MWCO)Sartocon Slice tangential flow filtration (TFF) system (Sartorius). Thefilter was first prepared by washing with a Hepes/saline solution (10 mMHepes, 130 mM NaCl, pH 7.0) and the permeate was sampled for pH andconductivity. Following concentration, the concentrated protein wassampled and tested for protein concentration and enzyme activity. A 6×buffer exchange was performed on the concentrated protein into the finalbuffer: 10 mM Hepes, 130 mM NaCl, pH 7.0. The concentrated protein waspassed though a 0.22 μm filter into a 20 L sterile storage bag. Theprotein was sampled and tested for protein concentration, enzymeactivity, free sulfhydryl groups, oligosaccharide profiling andosmolarity.

Tables 4 to 10 provide monitoring data related to each of thepurification steps described above, for each 3D35M cell lot.

TABLE 4 Q sepharose column data Parameter HUA0406C HUA0410C HUA0415CHUA0420C Load volume 10647 13524 12852 20418 (mL) Load Volume/ 3.1 4.94.5 7.3 Resin Volume ratio Column 2770 3840 2850 2880 Volume (mL) Eluatevolume 6108 5923 5759 6284 (mL) Protein Conc. 2.8 3.05 2.80 2.86 ofEluate (mg/mL) Eluate Enzyme 24493 26683 18321 21052 Assay (U/mL) EnzymeYield 65 107 87 76 (%)

TABLE 5 Phenyl Sepharose column data Parameter HUA0406C HUA0410CHUA0415C HUA0420C Volume Before Stock 5670 5015 5694 6251 SolutionAddition (mL) Load Volume (mL) 7599 6693 7631 8360 Column Volume (mL)9106 9420 9340 9420 Load Volume/Resin 0.8 0.71 0.82 0.89 Volume ratioEluate volume (mL) 16144 18010 16960 17328 Protein Cone of Eluate 0.40.33 0.33 0.38 (mg/mL) Eluate Enzyme Assay 8806 6585 4472 7509 (U/mL)Protein Yield (%) 41 40 36 37 Enzyme Yield (%) 102 88 82 96

TABLE 6 Amino Phenyl Boronate column data Parameter HUA0406C HUA0410CHUA0415C HUA0420C Load Volume (mL) 16136 17958 16931 17884 LoadVolume/Resin 2.99 3.15 3.08 2.98 Volume ratio Column Volume (mL) 54005700 5500 5300 Eluate volume (mL) 17595 22084 20686 19145 Protein Conc.of Eluate 0.0 0.03 0.03 0.04 (mg/mL) Protein Conc. of Filtered nottested 0.03 0.00 0.04 Eluate (mg/mL) Eluate Enzyme Assay 4050 2410 15234721 (U/mL) Protein Yield (%) 0 11 11 12 Enzyme Yield (%) not determined41 40 69

TABLE 7 Hydroxyapatite column data Parameter HUA0406C HUA0410C HUA0415CHUA0420C Volume Before Stock 16345 20799 20640 19103 Solution Addition(mL) Load Volume/Resin 10.95 13.58 14.19 12.81 Volume ratio ColumnVolume (mL) 1500 1540 1462 1500 Load volume (mL) 16429 20917 20746 19213Eluate volume (mL) 4100 2415 1936 2419 Protein Conc. of Eluate nottested 0.24 0.17 0.23 (mg/mL) Protein Conc. of Filtered NA NA 0.17 NAEluate (mg/mL) Eluate Enzyme Assay 14051 29089 20424 29826 (U/mL)Protein Yield (%) Not tested 93 53 73 Enzyme Yield (%) 87 118 140 104

TABLE 8 DV20 filtration data Parameter HUA0406C HUA0410C HUA0415CHUA0420C Start volume (mL) 4077 2233 1917 2419 Filtrate Volume (mL) 46023334 2963 3504 Protein Conc. of Filtrate 0.1 NA 0.09 NA (mg/mL) ProteinConc. of Filtered NA 0.15 0.09 0.16 Eluate (mg/mL) Protein Yield (%) nottested 93 82 101

TABLE 9 Final concentration data Parameter HUA0406C HUA0410C HUA0415CHUA0420C Start volume 4575 3298 2963 3492 (mL) Concentrate 562 407 237316 Volume (mL) Protein Conc. 0.9 1.24 1.16 1.73 of Concentrate (mg/mL)Protein Yield 111 102 103 98 (%)

TABLE 10 Buffer Exchange into Final Formulation data Parameter HUA0406CHUA0410C HUA0415C HUA0420C Start Volume (mL) 562 407 237 316 FinalVolume Buffer 594 516 310 554 Exchanged Concentrate (mL) Protein Conc.of 1.00 0.97 0.98 1.00 Concentrate (mg/mL) Protein Conc. of Filtered0.95 0.92 0.95 1.02 Concentrate (mg/mL) Protein Yield (%) 118 99 110 101

The purified and concentrated soluble rHuPH20 protein was ascepticallyfilled into sterile vials with 5 mL and 1 mL fill volumes. The proteinwas passed though a 0.22 μm filter to an operator controlled pump thatwas used to fill the vials using a gravimetric readout. The vials wereclosed with stoppers and secured with crimped caps. The closed vialswere visually inspected for foreign particles and then labeled.

Following labeling, the vials were flash-frozen by submersion in liquidnitrogen for no longer than 1 minute and stored at ≦−15° C. (−20±5° C.).Production and purification of soluble rHuPH20 using this method yieldedapproximately 400-700 mg soluble rHuPH20 with a specific activity of96,000 units/mg to 144,000 units/mg.

Example 4 Production of Gen2 Soluble rHuPH20

The Gen1 3D35M cell line described above was adapted to highermethotrexate levels to produce Gen2 clones. 3D35M cells were seeded fromestablished methotrexate-containing cultures into CD CHO mediumcontaining 8 mM GlutaMAX™-I and 1.0 μM methotrexate. The cells wereadapted to a higher methotrexate level by growing and passaging them 9times over a period of 46 days in a 37° C., 7% CO₂ humidified incubator.The amplified population of cells was cloned out by limiting dilution in96-well tissue culture plates containing medium with 2.0 μMmethotrexate. After approximately 4 weeks, clones were identified andclone 3E10B was selected for expansion. 3E10B cells were grown in CD CHOmedium containing 8 mM GlutaMAX™-I and 2.0 μM methotrexate for 20passages, with testing for cell viability by trypan blue staining andcounting with a hemocytometer, and enzyme activity by the microturbidityassay (described below in Example 9). A master cell bank (MCB) of the3E10B cell line was created and frozen and used for subsequent studies.

Amplification of the cell line continued by culturing 3E10B cells in CDCHO medium containing 8 mM GlutaMAX™-I and 4.0 μM methotrexate. Afterthe 12^(th) passage, cells were frozen in vials as a research cell bank(RCB). One vial of the RCB was thawed and cultured in medium containing8.0 μM methotrexate. After 5 days, the methotrexate concentration in themedium was increased to 16.0 μM, then 20.0 μM 18 days later. Cells fromthe 8^(th) passage in medium containing 20.0 μM methotrexate were clonedout by limiting dilution in 96-well tissue culture plates containing CDCHO medium containing 4 mM GlutaMAX™-I and 20.0 μM methotrexate. Clones1B3, 2B2 and 5C1 were identified 5-6 weeks later. Cells from the 9^(th)passage of 3D35M also were cloned out by limiting dilution in 96-welltissue culture plates with CD CHO medium containing 8 mM GlutaMAX™-I and20.0 μM methotrexate, and clones 1G11, 2E10 and 2G10 were identified.

Cells cultures of each of 1B3, 2B2, 5C1, 1G11, 2E10 and 2G10 were seededat a density of 4×10⁵ cells/mL in a volume of 50 mL in 250 mL shakerflasks. The cultures were allowed to grow and decline without additionalfeeds for 10-14 days to determine the growth rate and productivity ofthe cells. Samples were taken periodically and assayed for hyaluronidaseactivity (Tables 11 and 12).

TABLE 11 Hyaluronidase activity of 1B3, 2B2 and 5C1 cells SolublerHuPH20 Enzyme Hours post activity in cell culture (units) inoculation1B3 2B2 5C1 74 382 95 942 101 582 142 2287 144 955 169 1200 195 238 1611242 2139 265 3070 336 2252

TABLE 12 Hyaluronidase activity of 1B3, 2B2 and 2E10 cells SolublerHuPH20 Enzyme Hours post activity in cell culture (units) inoculation1B3 2B2 2E10 98 470 123 1179 143 2228 216 2814 290 2860 291 2542 3372992

Four cell lines (2B2, 2G10, 1G11 and 2E10) were compared in a study inwhich all were seeded at a density of 4×10⁵ cells/mL in a volume of 50mL in 250 mL shaker flask. All received 10% (v/v) feeds on day 8 and 5%feeds with feed media containing CD CHO medium supplemented with 50 g/Lglucose, 40 g/L yeast extract and 1.1 g/L sodium butyrate. The cellswere harvested on day 15. Samples were taken periodically and assayedfor soluble rHuPH20 enzymatic activity (Table 13).

TABLE 13 Hyaluronidase activity of 2E10, 1G11, 2G10 and 2B2 cellsSoluble rHuPH20 Enzyme Hours post activity in cell culture (units)inoculation 2E10 1G11 2G10 2B2 122 991 87 1688 124 878 194 2151 13872430 196 2642 285 6231 3831 7952 287 8822 364 5880 2955 11064 366 15684

In both the batch and fed-batch conditions, culture of 2B2 ellsexhibited higher enzymatic activity, although other cells (e.g. 2G10cells) also exhibited good enzyme productivity the 2B2 cell line was,therefore, selected for expansion in medium containing 20.0 μMmethotrexate. After the 11^(th) passage, 2B2 cells were frozen in vialsas a research cell bank (RCB).

Example 5 Enzymatic Activity of Soluble rHuPH20 Produced in 3E10B and2B2 Cells

Soluble rHuPH20 produced by 3E10B and 2B2 cells was assayed forenzymatic activity using the Microturbidity assay (Example 9). Frozenvials of 3E10B and 2B2 cells banks were thawed and the cells werecultured separately for two passages in growth medium (CD CHO mediumwith 8 mM GlutaMAX™-I and either 2.0 μM methotrexate for 3E10B cells, or20.0 μM methotrexate for 2B2 cells) in 37° C., 6% CO₂ in a humidifiedincubator. Cells were inoculated into 20 mL growth medium in 125 mLErlenmeyer flasks (Corning) at 5×10⁵cells/mL, and grown for 8 days in37° C., 6% CO² in a humidified incubator with a shaker platform rotatingat approximately 100 rpm. On days 8 and 10, the cultures received 5% v/vof feed medium containing CD CHO medium supplemented with 50 g/Lglucose, 50 g/L Yeast extract, and 2.2 g/L (20 mM) sodium butyrate toinitiate the production phase. The cultures were sampled during theproduction phase on day 8 (190 hours), day 10 (240 hours), day 14 (332hours), day 15 (258 hours), day 16 (382 hours) and day 18 (427 hours),and the viability was allowed to decline to zero. The samples were thenanalyzed for hyaluronidase activity.

Tables 14 and 15 set forth the viability (viable cell density (VCD) andpercentage viability) and activity (units/flask) of the 3E10B and 2B2cells at each time point. The enzyme activity of soluble rHuPH20produced by 2B2 cells was consistently higher than that produced by3E10B cells. For example, on day 8, the enzyme activity of solublerHuPH20 produced by 2B2 cells was 69% higher than that of produced by3E10B cells (2484 units/mL compared to 1469 units/mL). A similar trendwas observed throughout the production phase. The viability of the cellcultures declined at a similar rate. When the production rate of thecells was calculated, it was observed that 3E10B cells produced 0.23picograms soluble rHuPH20 per cell per day (pcd) on day 8 and 0.38 pcdon day 15. In comparison, 2B2 cells produced 0.46 picograms solublerHuPH20 pcd on day 8 and 0.69 pcd on day 15, which was 100% and 82%higher than production by 3E10B on days 8 and 15, respectively. A mastercell bank (MCB) of 2B2 cells was then prepared for subsequent studies.

TABLE 14 Viability and activity of Clone 3E10B Hours post Activityinoculation VCD % viability (Units/mL) Volume (mL) 0 5 99 0 20 190 79.896 1469 20 240 61.6 76 2388 20 332 16.8 22 5396 20 358 16.4 17 5628 20382 8.4 10 6772 20 427 0 0 6476 20 Total Activity (units) per flask(U/mL times volume (mL)): 129520

TABLE 15 Viability and activity of Clone 2B2 Hours post Activityinoculation VCD % viability (Units/flask) Volume (mL) 0 5 99 0 20 190 6894 2484 20 240 77.6 89 3532 20 332 32 38 8196 20 358 15.8 17 9680 20 3829.8 13 10788 20 427 0 0 10044 20 Total Activity (units) per flask (U/mLtimes volume (mL)): 200880

Example 6 Genetic Stability Testing of 2B2 Cells

A. Determination of Copy Number of the Nucleic Acid Region EncodingSoluble rHuPH20 in 2B2 Cells

The copy number of the nucleic acid region encoding soluble rHuPH20 in2B2 cells was determined by PCR. Total genomic DNA was extracted from2×10⁷ 2B2 cells from the MCB using a QIAamp DNeasy kit (Qiagen). GenomicDNA also was extracted from DG-44 CHO cells as a negative control. Thepurity of the extracted DNA was verified by agarose gel electrophoresisand UV spectrophotometry. To generate fragments of DNA (versus highmolecular weight DNA), the genomic DNA was sheared by sonication. Thisensured more accurate pipetting and template accessibility. Sixindependent dilutions of the genomic DNA (to amounts dilutionsequivalent to approximately 1000 cells/μl) from 2B2 and DG-44 cells wereprepared and analyzed in duplicate in two assays; a target assay, whichtargeted and amplified a sequence specific to the nucleic acid regionencoding soluble rHuPH20 plasmid DNA, and an endogenous control, whichtargeted and amplified GAPDH sequence. The endogenous control was usedas a normalizing the results. The target assay included a standard curvegenerated from a serial dilution of known amounts of the HZ24 plasmidmixed into DG-44 CHO genomic DNA. The endogenous control included astandard curve generated from serial dilutions of DG-44 CHO genomic DNAmixed with HZ24 plasmid DNA. The mammalian genome size was assumed to be3×10⁹ base pairs. Each assay included a negative control (no template)and a positive control (HZ24 plasmid DNA for the target assay and hostcell DNA for the endogenous control normalizing assay). The reactionswere prepared using the HZM3.P1 forward primer and the HZM3.P2 reverseprimer (set forth in SEQ ID NOS:52 and 53, respectively) and the HZM3probe (SEQ ID NO:54), which contained the fluorescent dyes 6FAM(6-carboxyfluorescin) and TAMRA (6-carboxytetramethylrhodamine). Thesamples were amplified using the Applied Biosystems Prism® 7900 SequenceDetection System with standard cycling conditions (50° C. for 2 minutes,95° C. 10 min, 95° C. 15 seconds and 60° C. for 1 min for 40 cycles).

The target gene copy numbers per cell were calculated as the ratio oftarget copies to normalized copies (GAPDH) for the six dilutions of 2B2genomic DNA. The Dixon Q Outlier statistics test was applied to the dataset. The copy number of the nucleic acid region encoding soluble rHuPH20plasmid in 2132 cells was found to be 206.61±8.35.

B. mRNA Sequence Analysis

The sequence of the PH20 mRNA generated from the HZ24 plasmid in 2B2cells was determined. RNA was extracted from 2×10⁷ 2B2 cells from theMCB using a RNeasy Mini Kit (Qiagen). The sample was treated with DNaseI to remove contaminating DNA, and the purity of the RNA was verified byagarose gel electrophoresis and UV spectrophotometry. A reversetranscription reaction using SuperScript™ Reverse Transcriptase(Invitrogen) and a control reaction lacking reverse transcriptase wasperformed using the extracted RNA and oligo d(t) and random primers. Theresulting cDNA products were then used as templates in PCRamplifications. Two different sets of primer pairs were used; AP01/AP03and AP10/AP12. AP01/AP03 was designed to amplify 1719 base pair region,while primer pair AP10/AP12 was designed to amplify a larger region(1811 base pairs) to obtain the reverse strand sequence of the 3′ end.Table 5 sets forth the sequences of the primers. Each PCR reactionincluded single primer controls, a negative control using the no reversetranscriptase control (described above) as template, and a positivecontrol with control primers and control template. The amplificationproducts were visualized by agarose gel electrophoresis and confirmed tobe of the expected size, then purified to remove excess primers anddNTPs by gel extraction or EXOSAP (USB).

The purified products were sequenced using the BigDye® Terminator v1.1Cycle Sequencing Kit (Applied Biosystems) and the primers set forth inTable 16. The sequence data were assembled and the derived consensussequence (SEQ ID NO:55) compared to the reference sequence usingSequencher software version 4.1.2 (Gene Code Corporation). A total of1449 base pairs of sequence data were generated. The sequence was foundto be identical to the reference sequence (SEQ ID NO:47) except for onebase pair difference at position 1131, which was observed to be athymidine (T) instead of the expected cytosine I. This is a silentmutation, with no effect on the amino acid sequence.

TABLE 16 Primers for PCR amplification and sequencing Primer nameSequence SEQ ID NO. AP01 TTCTCTCCACAGGTGTC 56 AP02 AAGATTTCCTTACAAGAC 57AP03 TGGCGAGAGGGGAAAGAC 58 AP04 CCATTTATTTGAACACTC 59 AP06CCGAACTCGATTGCGCAC 60 AP07 AGCCATTCCCAAATTGTC 61 AP08 CTCCCAGTTCAATTACAG62 AP09 CGTTAGCTATGGATCCTC 63 AP10 CGAGACAGAGAAGACTCTTGCG 64 AP12CATTCAACAGACCTTGCATTCC 65C. Southern Blot Analysis of 2B2 Cells

A Southern Blot analysis was performed on 2B2 cells to obtain astructure map. Total DNA was extracted from 1×10⁷ 2B2 cells and 1×10⁷control DG-44 cells using a Maxwell 16® system (Promega). The extractedDNA and a HZ24 plasmid control construct were evaluated for purity byagarose gel electrophoresis.

DNA from 2B2 cells, DG-44 cells and the HZ24 plasmid control weredigested with Spe I, Xba I, and a double digest using BamH I/Hind III.Another BamH I/Hind digest was performed on the HZ24 plasmid control andthe approximately 1.4 kb was purified by gel extraction andradioactively labeled with α-³²P to generate a labeled probe.Approximately 10 μg each of digested 2B2 DNA and DG-44 DNA, and 10 μgDG-44 DNA with 250 μg HZ24 plasmid DNA, was electrophoresed on anagarose gel. An image was take on the gel following electrophoresis,then a Southern blot transfer was performed. The nylon membrane washybridized with the labeled probe then washed at room temperature for 30minutes then twice at 55° C. for 30 minutes. An initial autoradiographwas exposed for 24 hours and visually inspected. It was determined thata longer exposure was needed, so a second autoradiograph was exposed for3 days for a darker exposure of the hybridized bands. After developingthe film, the bans were sized using an AlphaImager® (Alpha Innotech).

No hybridizing bands were observed for the DG-44 negative control digestand single hybridizing bands of expected sizes were observed in the HZ24digests (BamH I/Hind III digest: ˜1.4 kb; Spe I digest: ˜6.6 kb; Xba Idigest: ˜6.5 kb). There was one major hybridizing band of ˜7.7 kb andfour minor hybridizing bands (˜13.9, ˜6.6, ˜5.7 and ˜4.6 kb) observedusing 2B2 DNA digested with Spe I, one major hybridizing band of ˜5.0 kband two minor hybridizing bands (˜13.9 and ˜6.5 kb) observed using 2B2DNA digested with Xba I, and one single hybridizing band of ˜1.4 kbobserved using 2B2 DNA digested with BamH I/Hind III. The presence ofthe single ˜1.4 kb hybridizing band in the BamH I/Hind III indicatedthat there were no large sequence insertions or deletions within theprobed region. The results from the single Xba I and Spe I digestsindicate that there are multiple integration sites of the HZ24 plasmidin the genome if the 2B2 cells.

Example 7 Production of Gen2 Soluble rHuPH20 in 30 L Bioreactor CellCulture

Soluble rHuPH20 was produced and purified from 2B2 cells using a 36 Lbioreactor (30 L culture volume) to determine optimal processes forscale-up to a 400 L bioreactor (300 L culture volume). Four separate 36L bioreactor runs are detailed below in sections A to D.

A. Production and Characterization of Soluble rHuPH20 Lots 056-099 and056-100

A vial of 2B2 (1×10⁷ cells) was thawed and cultured at 37° C., 7% CO₂for 8 passages in CD CHO (Invitrogen, Carlsbad, Calif.) supplementedwith 20 μM methotrexate and 40 mL/L GlutaMAX™-I (Invitrogen), afterwhich it was expanded to 600 mL. One week later, the culture wasexpanded to 5 L in CD CHO medium supplemented with 40 mL/L GlutaMAX™-Iand no methotrexate. A 36 L bioreactor containing 25 L CD CHO mediumsupplemented with 1 L GlutaMAX™-I and 30 mL gentamicin sulfate wasinoculated with the 5 L culture at an initial seeding density of 3.6×10⁵cell/mL. The agitation set point of the bioreactor was set to 75 RPM;temperature: 37° C.; pH: 7.15; dissolved oxygen: 30%. The bioreactorreceived filtered air overlay and an air/oxygen/CO₂ sparge, ascontrolled by an Applikon controller.

The culture was fed 7 times throughout the bioreactor run, at 161, 184,237, 256, 280, 304 and 328 hours post inoculation. The feed media werefiltered into the bioreactor via peristaltic pump. The content of eachfeed media and the bioreactor feed parameters throughout the run areprovided in Tables 17 and 18, respectively.

TABLE 17 Feed Media formulations Feed Feed Feed Feed Feed Feed FeedComponent #1 #2 #3 #4 #5 #6 #7 CD CHO 1 L 1 L 1 L 1 L 1 L 1 L 1 L liquidmedium GlutaMAX ™-I 120 mL 80 mL 40 mL 40 mL 40 mL 30 mL 30 mL CD CHO48.6 g 24.3 g 0 0 0 0 0 AGT powder Yeastolate 150 mL 300 mL 300 mL 150mL 150 ml 0 0 Ultrafiltrate (200 g/L) Sodium 1.65 g 2.35 g 1.65 g 1.65 g1.65 g 1.65 g 1.65 g butyrate

TABLE 18 Bioreactor Parameters Hyalu- % ronidase Hours VCD viability pHUnits Vol (L) Glucose Feed 0 3.6 97 7.28 0 31 6000 — 15 6.2 94 7.45 11731 5780 — 44 11.3 97 7.15 290 31 5320 — 88 25.6 97 6.85 517 31 3430 —115 42.6 95 6.75 1132 31 2920 — 139 56.4 96 6.74 1320 31 2220 — 161 71.997 6.82 2296 31 520 Feed #1 184 83.9 96 6.81 2748 32 610 Feed #2 21382.7 96 6.87 3396 33 1190 — 237 80.5 89 7.21 4450 33 200 Feed #3 25662.3 71 7.03 4750 34 240 Feed #4 280 52.7 66 7.01 5030 35 600 Feed #5304 44.6 59 7.00 5970 36 560 Feed #6 328 33.3 47 7.00 7240 37 570 Feed#7 351 26.1 34 7.00 7360 37 250 —

The bioreactor was harvested and filtered through a system thatcontained a series of capsule filters (Sartorius) with pore sizes of 8μm, 0.65 μm, 0.45 μm and 0.22 μm, respectively. The harvest wasperformed using a peristaltic pump and completed in approximately 5hours, yielding approximately 32 L of harvested cell culture fluid(HCCF). The HCCF was supplemented with EDTA and Tris to finalconcentrations of 10 mM each, pH 7.6. The HCCF was then stored at 2-8°C. before being concentrated and subjected to a buffer exchange.

To concentrate the protein, a 2.5 ft² Millipore spiral wound cartridgewith a 30 kDa MWCO was first equilibrated in 150 mM NaCl, 10 mM Hepes,10 mM EDTA, pH 7.5. Fifteen L of HCCF was concentrated 15× to 1 L. Theconcentrate was 10× buffer exchanged with the 150 mM NaCl, 10 mM Hepes,10 mM EDTA, pH 7.5 buffer, and the retentate was filtered through a 0.2μm capsule into a 2 L storage bag, for a final volume of 1.1 L. Theretentate was then stored at 2-8° C.

The concentrated and buffer exchanged protein solution was then purifiedusing column chromatography through a Q Sepharose column, a phenylSepharose column, an Amino Phenyl column and a Hydroxyapatite column.The hyaluronidase units in the protein solution before and after eachchromatography step were assessed and used to determine the yield foreach step.

Briefly, a Q sepharose column with a 1.1 L column bed was sanitized with2.8 L 1.0 N NaOH and stored in 0.1 N NaOH prior to use. It was thencleaned with 2.5 L of 10 mM Hepes, 400 mM NaCl, pH 7.0, rinsed in 4.1 Lsterile water for injection (SWFI) and equilibrated with 2.5 L of 10 mMHepes, 25 mM NaCl, pH 7.0. The buffer exchanged protein (1 L at 170,160units/mL) was loaded onto the column. The flowthrough was 1.0 L at 479units/mL, indicating that nearly all of the product bound the resin. Thecolumn was washed with 4 L of 10 mM Hepes, 25 mM NaCl, pH 7.0, and 4.2 Lof 10 mM Hepes, 50 mM NaCl, pH 7.0. The product was then eluted in 3.0 Lof 10 mM Hepes, 400 mM NaCl, pH 7.0, yielding 3 L at 49,940 units/mL,and filtered through a 0.2 μm filter.

A Phenyl Sepharose column with a 2.1 L column bed was sanitized with 4.8L 1.0 N NaOH and stored in 0.1 N NaOH prior to use. It was then rinsedwith 5.0 L SWFI and cleaned with 4.6 L of 5 mM potassium phosphate, 0.5M ammonium sulfate and rinsed again with 6.8 L SWFI. The column was thenequilibrated in 5.5 L of 5 mM potassium phosphate, 0.5 M ammoniumsulfate. To the eluate from the Q Sepharose column, 10.3 mL of 1 Mpotassium phosphate monobasic, 10.3 mL 1 M potassium phosphate dibasicand 0.42 mL 1 mL CaCl₂ was added. This was then loaded onto the columnand the flow through and chase (1 L of 5 mM potassium phosphate, 0.5 mMammonium sulfate) were collected, yielding 7.4 L at 20,030 units/mL. Theproduct was filtered through a 0.2 μm filter.

An Amino Phenyl Boronate column with a 1.8 L column bed was sanitizedwith 4.5 L 1.0 N NaOH and stored in 0.1 N NaOH prior to use. It was thenrinsed with 3.9 L SWFI, cleaned with 4.2 L of 5 mM potassium phosphate,0.5 M ammonium sulfate and rinsed again with 4.0 L SWFI. The column wasthen equilibrated with 7.5 L of 5 mM potassium phosphate, 0.5 M ammoniumsulfate. The flow through material from the Phenyl Sepharose column wasloaded onto the Amino Phenyl Boronate column after being supplementedwith ammonium sulfate to a final concentration of 0.5°M. The column waswashed with 6.5 L of 5 mM potassium phosphate pH 7.0, then with 7.8 L of20 mM bicine, pH 9.0 then with 9.0 L of 20 mM bicine, 100 mM NaCl, pH9.0. The product was eluted with 4.8 L 50 mM Hepes, 100 mM NaCl, pH 7.0,resulting in 4.8 L at 22,940 units/mL, and filtered through a 0.2 μmfilter.

An Hydroxyapatite column with a 0.8 L column bed was sanitized with 2.7L 1.0 N NaOH and stored in 0.1 N NaOH prior to use. The column wasneutralized with 2.1 L of 200 mM potassium phosphate, pH 7.0 thenequilibrated in 2.2 L of 5 mM potassium phosphate, 100 mM NaCl. To theeluate from the Amino Phenyl Boronate column, 9.1 mL of 1 M potassiumphosphate monobasic, 9.1 mL 1 M potassium phosphate dibasic and 0.452 mL1 mL CaCl₂ was added. This was then loaded onto the column and the flowthrough was 4.5 L at 10 units/mL, indicating good binding of the solubleHuPH20. The column was washed with 3.3 L of 5 mM potassium phosphate,100 mM NaCl, 0.5 M ammonium sulfate, pH 7.0, then with 2.9 L of 10 mMpotassium phosphate, 100 mM NaCl, 0.5 M ammonium sulfate, pH 7.0. Theproduct was eluted with 1.0 L of 70 mM potassium phosphate, pH 7.0,resulting in 1 L at 130,000 units/mL, and filtered through a 0.2 μmfilter.

The purified product was concentrated using a 2.5 ft² Millipore 30 kDaMWCO cartridge that had been equilibrated in 130 mM NaCl, 10 mM Hepes,pH 7.0. The product was concentrated 74 mL, and buffer exchanged 10×with the 130 mM NaCl, 10 mM Hepes, pH 7.0 buffer then filtered through a0.2 μm filter. A₂₈₀ measurements were performed and indicated that theprotein concentration was 11.3 mg/mL. An additional 9.6 mL of 130 mMNaCl, 10 mM Hepes, pH 7.0 buffer was added to bring the proteinconcentration to 10 mg/mL (Lot 056-99). Ten mL of the 10 mg/mL proteinsolution was diluted in the buffer to yield a 1 mg/mL solution (Lot056-100. Both solutions were filtered through a 0.2 μm filter.

The formulated product was filled into 10 mL and 1 mL glass vials, thecombined total of which yielded 761 mg soluble rHuPH20. The vials werefrozen at −80° C. then transferred to −20° C. for storage. Lot 056-99and 056-100 were then characterized with respect to activity and purity.Lots 056-99 and 056-100 exhibited 1,350,786 units/mL and 129,982units/mL enzyme activity, and 130,00 units/mg and 124,00 units/mgspecific activity (calculated from enzyme activity and proteinconcentration). The purity of the soluble rHuPH20 samples was determinedby SDS-PAGE, IsoElectric Focusing (IEF), reverse phase high pressureliquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) andanion-exchange chromatography. As determined by RP-HPLC, purity of thetwo Lots was observed to be approximately 95%. As determined by SEC,purity of the two Lots was observed to be approximately 99%. Endotoxinlevels were shown to be <0.5 EU/mL and 0.1 EU/mL for lots 056-99 and056-100, respectively. Osmolarity was measured to be 271 mOsm/kg and 291mOsm/kg for lots 056-99 and 056-100, respectively.

B. Modifications to Increase Soluble rHuPH20 Production: BioreactorBatch 2B2-20K.5

Modifications were made to the method described above in section A.These modifications were intended to increase the product yield andimprove the efficiency and scalability of manufacturing. Themanufacturing steps described below include thaw of frozen cells fromthe research cell bank, expansion of cells in continuous culture,operation of fed-batch bioreactor system, harvest and clarification ofcell culture fluid, and concentration and buffer exchange of bulkproduct. The modifications include, for example, the addition ofrecombinant human insulin to the bioreactor medium to increase thegrowth rate and product expression levels of the cells. Also, the numberof feeds has been reduced from 7 to 5. Other modifications of themethods described above also were made.

A vial of 2B2 (1×10⁷ cells) was thawed and cultured in CD CHO(Invitrogen, Carlsbad, Calif.) supplemented with 20 μM methotrexate and40 mL/L GlutaMAX™-I (Invitrogen), after which it was expanded to 100 mL,450 ml then to 4.5 L in CD CHO medium supplemented with 40 mL/LGlutaMAX™-I and no methotrexate. A 36 L bioreactor containing 20 L CDCHO medium supplemented with 800 L GlutaMAX™-I, 30 mL gentamicin sulfateand 100 mg recombinant human insulin was inoculated with 3.6 L 2B2culture at an initial seeding density of 4.3×10⁵ cell/mL. The agitationset point of the bioreactor was set to 80 RPM; temperature: 37° C.; pH:7.15; dissolved oxygen: 25%. The bioreactor received filtered airoverlay and an air/oxygen/CO₂ sparge, as controlled by an Applikoncontroller.

The culture was fed 5 times throughout the 13 day bioreactor run, at117, 143, 196, 235, and 283 hours post inoculation. The feed media werefiltered into the bioreactor via peristaltic pump. The content of eachfeed media and the bioreactor feed parameters throughout the run areprovided in Tables 19 and 20, respectively.

TABLE 19 Feed Media formulations Initial bioreactor Feed Feed Feed FeedComponent medium Feed #1 #2 #3 #4 #5 CD CHO 12 L 0 0 0 0 0 liquid mediumGlutaMAX ™-I 800 mL 120 mL 80 mL 40 mL 40 mL 40 mL CD CHO 194.4 g 97.2 g48.6 g 24.3 g 24.3 g 24.3 g AGT powder SWFI 8 L 800 mL 900 mL 700 mL 700mL 700 mL Yeastolate 0 0 0 200 mL 200 mL 200 mL Ultrafiltrate (200 g/L)Dextrose 0 30 g 60 g 40 g 40 g 40 g Gentamicin 30 mL 0 0 0 0 0rHuInsulin 25 mL 0 0 0 0 0 Sodium 0 0 0 2.2 g 1.1 g 1.1 g butyrate

TABLE 20 Bioreactor Parameters Hyalu- % ronidase Hours VCD viability pHUnits Vol (L) Glucose Feed 0 4.3 98 7.28 0 25 8820 — 55 17.1 99 7.07 58025 4950 — 94 40.6 99 6.77 1059 25 3800 — 117 57.5 99 6.76 1720 25 2310Feed #1 143 88.8 99 6.75 3168 26 2770 Feed #2 167 93.7 99 6.80 6982 273830 — 196 96.2 97 6.89 4560 27 2060 Feed #3 222 78.9 85 6.83 4920 282720 — 235 80 76 6.81 5670 28 1870 Feed #4 260 54.3 65 6.76 5865 29 2930— 283 38.7 44 6.73 6540 29 1880 Feed #5 308 37.3 39 6.78 8460 29 2400 —313 33.7 34 6.78 8190 29 2300 —

The bioreactor was harvested and filtered through a system thatcontained a series of Millipore Pod filters DOHC (0.5 m²) and A1HCstacks, which contain layers of graded-pore-size diatomaceous earth,followed by final filtration through capsule filter (Sartorius Sartobran300) into a 50 L storage bag. The harvest was performed using aperistaltic pump and completed in approximately 2 hours, yieldingapproximately 30 L of harvested cell culture fluid (HCCF). Twenty-eightL HCCF was supplemented with EDTA and Tris to final concentrations of 10mM each, and a pH 7.5. The remaining 2 1 HCCF was left withoutTris/EDTA, to assess the effect of adding Tris/EDTA on theconcentration/buffer exchange step. The HCCF was then stored at 2-8° C.before being concentrated and subjected to a buffer exchange.

To concentrate the protein, a 0.1 m² Millipore Pellicon 2 biomax Ascreen cassette with a 30 kDa MWCO was first equilibrated in 20 mMNa₂SO₄, 10 mM Tris, pH 7.5. 2 L of HCCF with and without Tris/EDTA wasconcentrated 10× and buffer exchanged 10× with the 20 mM Na₂SO₄, 10 mMTris, pH 7.5 buffer. The protein levels were measured by absorbance atA₂₆₀. The remaining HCCF (approximately 26.5 L) was then concentratedand subjected to buffer exchange. Two 0.1 m² Millipore Pellicon 2 biomaxA screen cassettes with a 30 kDa MWCO were assembled in the TFF systemand equilibrated in 20 mM Na₂SO₄, 10 mM Tris, pH 7.5. The HCCF wasconcentrated approximately 10× to 2.5 L, and buffer exchanged 10× with20 mM Na₂SO₄, 10 mM Tris, pH 7.5. The retentate was filtered through a0.2 μm vacuum filter into 1 L and 500 mL storage bags, for a finalvolume of 2.6 L. The retentate was then stored at 2-8° C. Samples takenduring the concentration and buffer exchange process were analyzed byRP-HPLC to determine the effect of the addition of Tris/EDTA to thesample. It was observed that the addition of Tris/EDTA facilitated amore efficient processing step.

C. Production and Characterization of Soluble rHuPH20 Lots 056-122 and056-123 (Bioreactor Batch 2B2-20K.6).

Modifications described above in section C were incorporated into themanufacturing steps to produce and purify two lots of soluble rHuPH20;Lots 056-122 and 056-123. The process described below include thaw offrozen cells from the research cell bank HZ24-2B2; expansion of cells incontinuous culture; operation of 36 L fed-batch bioreactor system at the30 L scale; cell removal, clarification, and buffer exchange of bulkproduct; 4-step column chromatography; and formulation, fill, and finishoperations.

A vial of 2B2 (1×10⁷ cells) was thawed and cultured at 37° C., 7% CO₂ inCD CHO (Invitrogen, Carlsbad, Calif.) supplemented with 20 μMmethotrexate and 40 mL/L GlutaMAX™-I (Invitrogen), after which it wasexpanded to 400 mL then 4.4 L in CD CHO medium supplemented with 40 mL/LGlutaMAX™-I and no methotrexate. A 36 L bioreactor (Bellco 1964 series)containing 20 L CD CHO medium supplemented with 800 L GlutaMAX™-I, 100mg recombinant human insulin and 30 mL gentamicin sulfate was inoculatedwith the 4 L culture at an initial seeding density of 4.9×10⁵ cell/mL.The agitation set point of the bioreactor was set to 80 RPM;temperature: 37° C.; pH: 7.15; dissolved oxygen: 25%. The bioreactorreceived filtered air overlay and an air/oxygen/CO₂ sparge, ascontrolled by an Applikon ADI 1030 controller.

The culture was fed 4 times throughout the 13 day bioreactor run, at127, 163, 208 and hours post inoculation. The feed media were filteredinto the bioreactor via peristaltic pump. The temperature setpoint ofthe bioreactor was reduced from 37° C. to 36.5° C. on day 7, to 36.0° C.on day 9 and finally to 35.5° C. on day 11. The content of each feedmedia and the bioreactor feed parameters throughout the run are providedin Tables 21 and 22, respectively.

TABLE 21 Feed Media formulations Initial bioreactor Component mediumFeed #1 Feed #2 Feed #3 Feed #4 CD CHO AGT powder 0 97.2 g 48.6 g 24.3 g24.3 g (Invitrogen; part #: 10743-029; lot # 1366333) CD CHO AGT powder267.3 g 0 0 0 0 (Invitrogen; part #: 10743-029; lot # 1320613) CD CHOAGT powder 218.7 g 0 0 0 0 (Invitrogen; part #: 12490-017; lot #1300803) SWFI 20 L 700 mL 700 mL 600 mL 600 mL GlutaMAX ™-I 800 mL 160mL 80 mL 60 mL 40 mL (Invitrogen) Yeastolate Ultrafiltrate 0 100 mL 200mL 300 mL 300 mL (Invitrogen; 200 g/L) Dextrose (D-glucose) 0 40 g 40 g60 g 40 g Gentamicin 30 mL 0 0 0 0 rHuInsulin 100 mg 40 mg 0 0 0 Sodiumbutyrate 0 0 1.1 g 2.2 g 1.1 g

TABLE 22 Bioreactor Parameters Viable cell density Hyalu- (×10⁵ %ronidase Vol Hours cells/mL) viability pH Units (L) Glucose Feed 0 4.992 7.26 79 25 7780 — 24 9.2 95 7.21 141 25 6060 — 48 17.3 97 7.13 243 255280 — 72 33 99 6.82 407 25 3910 — 98 49.3 99 6.77 658 25 3200 — 127 6798 6.83 1296 25 1610 Feed #1 144 88.1 98 6.78 1886 26 2860 — 163 92.4 996.89 2439 26 1680 Feed #2 192 91 97 6.85 3140 27 1480 — 208 92.7 96 6.913188 27 230 Feed #3 235 70 76 6.86 5118 28 1940 — 261 63 61 6.84 5862 28280 Feed #4 291 36.4 45 6.76 7072 29 1570 — 307 29.3 32 6.81 8160 291250 Harvest

The bioreactor was harvested and filtered through a system thatcontained a series of Millipore Pod filters DOHC (0.5 m²) and A1HCstacks (0.1 m²), which contain layers of graded-pore-size diatomaceousearth, followed by final filtration through capsule filter (SartoriusSartobran 300) into 20 L storage bags. The harvest was performed using aperistaltic pump and completed in approximately 1 hour, yieldingapproximately 34 L of harvested cell culture fluid (HCCF). This includesthe 29 L bioreactor volume plus approximately 5 L PBS chase. The HCCFwas supplemented with EDTA and Tris to final concentrations of 10 mMeach, and a final pH of 7.5. The HCCF was then stored at 2-8° C. beforebeing concentrated and subjected to a buffer exchange.

To concentrate the protein, a 7.0 ft² Sartorius Sartocon 2 crossflowcassette with a 30 kDa MWCO was first equilibrated in 20 mM Na₂SO₄, 10mM Tris, pH 7.5. 34 L of HCCF was concentrated 10× to 3 L and bufferexchanged 10× with the 20 mM Na₂SO₄, 10 mM Tris, pH 7.5 buffer. Theretentate was filtered through a 0.2 μm capsule filter into a 5 Lstorage bags for a final volume of 3.0 L. The retentate was then storedat 2-8° C.

The concentrated and buffer exchanged protein solution was then purifiedusing column chromatography through a Q Sepharose column, a phenylSepharose column, an Amino Phenyl column and a Hydroxyapatite column.The hyaluronidase units in the protein solution before and after eachchromatography step were assessed and used to determine the yield foreach step.

Briefly, a Q sepharose column with a 1.1 L column bed, diameter 7 cm,height 28 cm was sanitized with 2.1 L 1.0 N NaOH and stored in 0.1 NNaOH prior to use. It was then cleaned with 2.5 L of 10 mM Hepes, 400 mMNaCl, pH 7.0, rinsed in 4.5 L sterile water for injection (SWFI) andequilibrated with 4.3 L of 10 mM Hepes, 25 mM NaCl, pH 7.0. The bufferexchanged protein (3 L at 94,960 hyaluronidase units/mL) was loaded ontothe column. The flowthrough and first wash was 5830 mL at 75hyaluronidase units/mL, indicating that nearly all of the product(99.8%) bound the resin. The column was washed with 4.2 L of 10 mMHepes, 25 mM NaCl, pH 7.0, and 4.6 L of 10 mM Hepes, 50 mM NaCl, pH 7.0.The product was then eluted in 2.9 L of 10 mM Hepes, 400 mM NaCl, pH7.0, yielding 2880 mL at 96,080 units/mL, and filtered through a 0.2 μmfilter.

A Phenyl Sepharose column with a 2.2 L column bed (height 28 cm,diameter 10 cm) was sanitized with 5.0 L 1.0 N NaOH and stored in 0.1 NNaOH prior to use. It was then rinsed with 4.5 L SWFI and cleaned with4.6 L of 5 mM potassium phosphate, 0.5 M ammonium sulfate and rinsedagain with 6.8 L SWFI. The column was then equilibrated in 4.6 L of 5 mMpotassium phosphate, 0.5 mM ammonium sulfate. To the eluate from the QSepharose column, 9.6 mL of 1 M potassium phosphate monobasic, 9.6 mL 1M potassium phosphate dibasic and 0.4 mL 1 mL CaCl₂ was added. This wasthen loaded onto the column and the flow through and chase (5 mMpotassium phosphate, 0.5 mM ammonium sulfate) were collected, yielding6905 mL at 36,280 units/mL. The product was filtered through a 0.2 μmfilter.

An Amino Phenyl Boronate column with a 2.2 L column bed (height 29 cm,diameter 10 cm) was sanitized with 3.8 L 1.0 N NaOH and stored in 0.1 NNaOH prior to use. It was then rinsed with 5.0 L SWFI, cleaned with 5.0L of 5 mM potassium phosphate, 0.5 M ammonium sulfate and rinsed againwith 5.0 L SWFI. The column was then equilibrated with 5.0 L of 5 mMpotassium phosphate, 0.5 M ammonium sulfate. The flow through materialfrom the Phenyl Sepharose column was loaded onto the Amino PhenylBoronate column. The column was washed with 9.9 L of 5 mM potassiumphosphate, 0.5M ammonium sulfate, pH 7.0, then with 9.7 L of 20 mMbicine, 0.5 M ammonium sulfate, pH 9.0 then with 9.9 L of 20 mM bicine,100 mM NaCl, pH 9.0. The product was eluted with 5.0 L 50 mM Hepes, 100mM NaCl, pH 7.0, resulting in 4460 mL at 48,400 units/mL, and filteredthrough a 0.2 μm filter.

A Hydroxyapatite column with a 1.1 L column bed (diameter 7 cm, height28 cm) was sanitized with 2.7 L 1.0 N NaOH and stored in 0.1 N NaOHprior to use. The column was neutralized with 2.1 L of 200 mM potassiumphosphate, pH 7.0, then equilibrated in 2.2 L of 5 mM potassiumphosphate, 100 mM NaCl. To the eluate from the Amino Phenyl Boronatecolumn, 11.2 mL of 1 M potassium phosphate monobasic, 11.2 mL 1 Mpotassium phosphate dibasic and 0.45 mL 1 mL CaCl₂ was added. This wasthen loaded onto the column and subsequently washed with 3.5 L of 5 mMpotassium phosphate, 100 mM NaCl, 0.5 M ammonium sulfate, pH 7.0, thenwith 3.5 L of 10 mM potassium phosphate, 100 mM NaCl, 0.5 M ammoniumsulfate, pH 7.0. The product was eluted with 1.4 L of 70 mM potassiumphosphate, pH 7.0, resulting in 1260 mL at 152,560 units/mL, andfiltered through a 0.2 μm filter.

The purified product was concentrated using a 0.05 ft² Millipore 30 kDaMWCO cartridge that had been equilibrated in 130 mM NaCl, 10 mM Hepes,pH 7.0. The product was concentrated from 1250 mL at 1.04/mg/mL to 120mL and buffer exchanged 10× with the 130 mM NaCl, 10 mM Hepes, pH 7.0buffer then filtered through a 0.2 μm filter. A₂₈₀ measurements wereperformed and indicated that the soluble rHuPH20 concentration of theremaining 118 ml was 11.45 mg/mL. An additional 17 mL of 130 mM NaCl, 10mM Hepes, pH 7.0 buffer was added to bring the protein concentration to10 mg/mL (Lot 056-122). Ten mL of the 10 mg/mL protein solution wasdiluted in the buffer to yield a 1 mg/mL solution (Lot 056-123. Bothsolutions were filtered through a 0.2 μm filter.

The formulated product was filled into 10 mL and 1 mL glass vials, thecombined total of which yielded 1308 mg soluble rHuPH20. The vials werefrozen at −80° C. then transferred to −20° C. for storage. Lot 056-122and 056-123 were then characterized with respect to activity and purity.Lots 056-122 and 056-123 exhibited 1,376,992 units/mL and 129,412units/mL enzyme activity, and 136,900 units/mg and 124,400 units/mgspecific activity (calculated from enzyme activity and proteinconcentration). The purity of the soluble rHuPH20 samples was determinedby SDS-PAGE, IsoElectric Focusing (IEF), reverse phase high pressureliquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) andanion-exchange chromatography. As determined by RP-HPLC, purity of thetwo Lots was observed to be approximately 96.2%. As determined by SEC,purity of the two Lots was observed to be greater than 99%. Endotoxinlevels were shown to be <0.8 EU/mL and 0.09 EU/mL for lots 056-122 and056-123, respectively. Osmolarity was measured to be 265 mOsm/kg and 256mOsm/kg for lots 056-122 and 056-123, respectively. The pH of each was7.2.

D. Reproducibility of the Production Process of Gen 2 Soluble rHuPH20 in30 L Bioreactor Cell Culture

The process described above for Batch 2B2-20K.6 was used for asubsequent batch to demonstrate the reproducibility of the process. Theprocess was modified slightly by the incorporation of a viralinactivation step immediately prior to the column chromatography steps.

A vial of 2B2 cells (1×10⁷ cells) was thawed and cultured at 37° C., 7%CO₂ for 7 passages in CD CHO (Invitrogen, Carlsbad, Calif.) supplementedwith 20 μM methotrexate and 40 mL/L GlutaMAX™-I (Invitrogen), afterwhich it was expanded to 400 mL then 4.4 L in CD CHO medium supplementedwith 40 mL/L GlutaMAX™-I and no methotrexate. A 36 L bioreactor (Bellco1964 series) containing 20 L CD CHO medium supplemented with 800 mLGlutaMAX™-I, 100 mg recombinant human insulin and 300 mg gentamicinsulfate was inoculated with 3 L culture at an initial seeding density of4.7×10⁵ cell/mL. The agitation set point of the bioreactor was set to 80RPM; temperature: 37° C.; pH: 7.15; dissolved oxygen: 25%. Thebioreactor received filtered air overlay and an air/oxygen/CO₂ sparge,as controlled by an Applikon ADI 1030 controller.

The culture was fed 4 times throughout the 13 day bioreactor run, at127, 163, 208 and hours post inoculation. The feed media were filteredinto the bioreactor via peristaltic pump. The temperature setpoint ofthe bioreactor was reduced from 37° C. to 36.5° C. on day 7, to 36.0° C.on day 9 and finally to 35.5° C. on day 11. The content of each feedmedia and the bioreactor feed parameters throughout the run are providedin Tables 23 and 24, respectively.

TABLE 23 Feed Media formulations Initial bioreactor Component mediumFeed #1 Feed #2 Feed #3 Feed #4 CD CHO liquid 20 L 0 0 0 0 medium(Invitrogen) CD CHO AGT powder 0 g 97.2 g 48.6 g 24.3 g 24.3 g SWFI 0700 mL 700 mL 600 mL 600 mL GlutaMAX ™-I 800 mL 160 mL 80 mL 60 mL 40 mL(Invitrogen) Yeastolate Ultrafiltrate 0 100 mL 200 mL 300 mL 300 mL(Invitrogen; 200 g/L) Dextrose (D-glucose) 0 40 g 40 g 60 g 50 gGentamicin 300 mg 0 0 0 0 rHuInsulin 100 mg 40 mg 0 0 0 Sodium butyrate0 0 1.1 g 2.2 g 1.1 g

TABLE 24 Bioreactor Parameters Viable cell density Hyalu- (×10⁵ %ronidase Vol Hours cells/mL) viability pH Units (L) Glucose Feed 0 4.798 7.28 113 24 8200 — 24 8.9 98 7.23 202 24 6160 — 50 19.3 97 7.15 33224 5480 — 76 36.7 99 6.85 680 24 3620 — 120 73.6 99 6.76 1619 24 2100Feed #1 145 84.3 99 6.75 2842 25 2660 — 165 98.8 99 6.87 3756 25 840Feed #2 190 95.3 99 6.85 4773 26 1330 — 201 105 97 6.90 5484 26 270 Feed#3 214 95.9 93 6.82 6344 27 2590 — 242 81.2 81 6.75 7890 27 1350 Feed #4268 51.9 48 6.65 10398 28 2500 — 287 38.4 41 6.70 11864 28 2170 — 30831.6 31 6.66 12864 28 1850 Harvest

The bioreactor was harvested and filtered through a system thatcontained a series of Millipore Pod filters DOHC (0.5 m²) and A 1 HCstacks (0.1 m²), which contain layers of graded-pore-size diatomaceousearth, followed by final filtration through capsule filter (SartoriusSartobran 300) into 20 L storage bags. The harvest was performed usingaperistaltic pump and completed in approximately 75 minutes, yieldingapproximately 30 L of harvested cell culture fluid (HCCF). This includesthe 28 L bioreactor volume plus approximately 2 L PBS chase. The HCCFwas supplemented with EDTA and Tris to final concentrations of 10 mMeach, and a final pH of 7.5. The HCCF was then stored at 2-8° C. beforebeing concentrated and subjected to a buffer exchange.

To concentrate the protein, a Sartorius Slice system with 3×1.0 ft²Sartocon Slice crossflow cassettes (30 kDa MWCO) was first equilibratedin 20 mM Na₂SO₄, mM Tris, pH 7.5. Thirty liters of HCCF was concentrated10× to 3 L and buffer exchanged 10× with the 20 mM Na₂SO₄, 10 mM Tris,pH 7.5 buffer. The average flux rate during the concentration was 115mL/minute and the average transmembrane pressure was 16 psig. Theaverage flux rate during the diafiltration was 150 mL/minute and theaverage transmembrane pressure was 15 psig. The retentate was filteredthrough a 0.2 μM vacuum filter systems into a 5 L storage bags for afinal volume of 3.0 L. The retentate was then stored at 2-8° C.

Viral inactivation was performed by mixing 235 mL of a filtered solutionof 10% w/w Triton X-100, 35 w/w Tri-butyl phosphate in SWFI with 2.15 Lof room-temperature concentrated and buffer exchanged protein in a glassspinner flask stirring at 30-40 rpm. After 45 minutes, the proteinsolution was loaded onto the Q sepharose column (as described below).The loading took 24 minutes, which resulted in a total exposure time tothe detergent solution of 69 minutes.

The Q sepharose column with a 1.1 L column bed, diameter 7 cm, height 28cm was sanitized with 2.1 L 1.0 N NaOH and stored in 0.1 N NaOH prior touse. It was then cleaned with 2.5 L of 10 mM Hepes, 400 mM NaCl, pH 7.0,rinsed in 4.5 L sterile water for injection (SWFI) and equilibrated with4.5 L of 10 mM Hepes, 25 mM NaCl, pH 7.0. The buffer exchanged, viralinactivated protein (2385 mL at 133,040 hyaluronidase units/mL) wasloaded onto the column. The column was washed with 4.5 L of 10 mM Hepes,25 mM NaCl, pH 7.0, and 4.5 L of 10 mM Hepes, 50 mM NaCl, pH 7.0. Theproduct was then eluted in 2.5 L of 10 mM Hepes, 400 mM NaCl, pH 7.0,yielding 2500 mL at 133,680 units/mL, and filtered through a 0.2 μmfilter.

A Phenyl Sepharose column with a 2.2 L column bed (height 28 cm,diameter 10 cm) was sanitized with 5.0 L 1.0 N NaOH and stored in 0.1 NNaOH prior to use. It was then rinsed with 6.0 L SWFI and equilibratedin 4.6 L of 5 mM potassium phosphate, 0.5 mM ammonium sulfate. To elutethe column, 9.6 mL of 1 M potassium phosphate monobasic, 9.6 mL 1 Mpotassium phosphate dibasic and 0.4 mL 1 mL CaCl₂ was added. This wasthen loaded onto the column and the flow through and chase (5 mMpotassium phosphate, 0.5 mM ammonium sulfate) were collected, yielding6450 mL at 43,840 units/mL. The product was filtered through a 0.2 μmfilter.

An Amino Phenyl Boronate column with a 2.2 L column bed (height 29 cm,diameter 10 cm) was sanitized with 3.5 L 1.0 N NaOH and stored in 0.1 NNaOH prior to use. It was then rinsed with 5.0 L SWFI and equilibratedwith 9.0 L of 5 mM potassium phosphate, 0.5 M ammonium sulfate. The flowthrough material from the Phenyl Sepharose column was loaded onto theAmino Phenyl Boronate column. The column was washed with 9.9 L of 5 mMpotassium phosphate, 0.5 M ammonium sulfate, pH 7.0, then with 9.9 L of20 mM bicine, 0.5 M ammonium sulfate, pH 9.0. The product was elutedwith 4.4 L 50 mM Hepes, 100 mM NaCl, pH 7.0, yielding 4389 mL at 33,840units/mL, and filtered through a 0.2 μm filter.

A Hydroxyapatite column with a 1.1 L column bed (diameter 7 cm, height28 cm) was sanitized with 2.1 L 1.0 N NaOH and stored in 0.1 N NaOHprior to use. The column was neutralized with 3.6 L of 200 mM potassiumphosphate, pH 7.0, then equilibrated in 3.2 L of 5 mM potassiumphosphate, 100 mM NaCl. To the eluate from the Amino Phenyl Boronatecolumn, 11 mL of 1 M potassium phosphate monobasic, 11 mL 1 M potassiumphosphate dibasic and 0.44 mL 1 mL CaCl₂ was added. This was then loadedonto the column and subsequently washed with 4.8 L of 5 mM potassiumphosphate, 100 mM NaCl, 0.5 M ammonium sulfate, pH 7.0, then with 3.8 Lof 10 mM potassium phosphate, 100 mM NaCl, 0.5 M ammonium sulfate, pH7.0. The product was eluted with 1.5 L of 70 mM potassium phosphate, pH7.0, resulting in 1500 mL at 114,320 units/mL, and filtered through a0.2 μm filter.

The purified product was concentrated using a 0.05 ft² Millipore 30 kDaMWCO cartridge that had been equilibrated in 130 mM NaCl, 10 mM Hepes,pH 7.0. The product was concentrated from 1500 mL at 0.961 mg/mL to 125mL and buffer exchanged 10× with the 130 mM NaCl, 10 mM Hepes, pH 7.0buffer then filtered through a 0.2 μm filter. A₂₈₀ measurements wereperformed and indicated that the protein concentration of the remaining122 ml was 11.431 mg/mL. An additional 17.5 mL of 130 mM NaCl, 10 mMHepes, pH 7.0 buffer was added to bring the protein concentration to 10mg/mL (Lot 056-135). Ten mL of the 10 mg/mL protein solution was dilutedin the buffer to yield a 1 mg/mL solution (Lot 056-136). Both solutionswere filtered through a 0.2 μm filter.

The formulated product was filled into 5 mL and 1 mL glass vials, thecombined total of which yielded 1324 mg soluble rHuPH20. The vials werefrozen at −80° C. then transferred to −20° C. for storage. Lot 056-135and 056-136 were then characterized with respect to activity and purity.Lots 056-135 and 056-136 exhibited 1,301,010 units/mL and 127,661units/mL enzyme activity, and 121,600 units/mg and 127,700 units/mgspecific activity (calculated from enzyme activity and proteinconcentration). The purity of the soluble rHuPH20 samples was determinedby SDS-PAGE, IsoElectric Focusing (IEF), reverse phase high pressureliquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) andanion-exchange chromatography. As determined by RP-HPLC, purity of thetwo Lots was observed to be between 93.5% and 93.7%. As determined bySEC, purity of the two Lots was observed to be greater than 99%.Endotoxin levels were shown to be <0.84 EU/mL and 0.09 EU/mL for lots056-135 and 056-136, respectively. Osmolarity was measured to be 255mOsm/kg and 260 mOsm/kg for lots 056-135 and 056-136, respectively. ThepH of each was 7.2.

Example 8

A. Production of Gen2 Soluble rHuPH20 in 300 L Bioreactor Cell Culture

The production and purification methods detailed in Example 7, above,were scaled-up for production using a 400 L bioreactor. A vial of 2B2cells (1×10⁷ cells) was thawed and expanded from shaker flasks through36 L spinner flasks in CD CHO (Invitrogen, Carlsbad, Calif.)supplemented with 20 μM methotrexate and 8 mM GlutaMAX™-I (Invitrogen).Briefly, the a vial of cells was thawed in a 37° C. water bath, mediawas added and the cells were centrifuged. The cells were re-suspended ina 125 mL shake flask with 20 mL of fresh media and placed in a 37° C.,7% CO₂ incubator. The cells were expanded up to 40 mL in the 125 mLshake flask. When the cell density reached greater than 1.5×10⁶cells/mL, the culture was expanded into a 125 mL spinner flask in a 100mL culture volume. The flask was incubated at 37° C., 7% CO₂. When thecell density reached greater than 1.5×10⁶ cells/mL, the culture wasexpanded into a 250 mL spinner flask in 200 mL culture volume, and theflask was incubated at 37° C., 7% CO₂. When the cell density reachedgreater than 1.5×10⁶ cells/mL, the culture was expanded into a 1 Lspinner flask in 800 mL culture volume and incubated at 37° C., 7% CO₂.When the cell density reached greater than 1.5×10⁶ cells/mL, the culturewas expanded into a 6 L spinner flask in 5000 mL culture volume andincubated at 37° C., 7% CO₂. When the cell density reached greater than1.5×10⁶ cells/mL the culture was expanded into a 36 L spinner flask in32 L culture volume and incubated at 37° C., 7% CO₂.

A 400 L reactor was sterilized by steam at 121° C. for 30 minutes and230 mL of CD CHO media supplemented with 8 mM GlutaMAX™-I and 5 mg/LrHuInsulin was added. Before use, the reactor was checked forcontamination. Approximately 30 L cells were transferred from the 36 Lspinner flasks to the 400 L bioreactor (Braun) at an inoculation densityof 4.0×10⁵ viable cells per ml and a total volume of 260 L. Parameterswere temperature setpoint, 37° C.; Impeller Speed 40-55 RPM; VesselPressure: 3 psi; Air Sparge 0.5-1.5 L/Min.; Air Overlay: 3 L/min. Thereactor was sampled daily for cell counts, pH verification, mediaanalysis, protein production and retention. Also, during the runnutrient feeds were added. At 120 hrs (day 5), 10.4 L of Feed #1 Medium(4×CD CHO+33 g/L Glucose+160 mL/L GlutaMAX™-I+16.6 g/L Yeastolate+33mg/L rHuInsulin) was added. At 168 hours (day 7), 10.8 L of Feed #2(2×CD CHO+33 g/L Glucose+80 mL/L GlutaMAX™-I+33.4 g/L Yeastolate+0.92g/L Sodium Butyrate) was added, and culture temperature was changed to36.5° C. At 216 hours (day 9), 10.8 L of Feed #3 (1×CD CHO+50 g/LGlucose+50 mL/L GlutaMAX™-I+50 g/L Yeastolate+1.80 g/L Sodium Butyrate)was added, and culture temperature was changed to 36° C. At 264 hours(day 11), 10.8 L of Feed #4 (1× CD CHO+33 g/L Glucose+33 mL/LGlutaMAX™-I+50 g/L Yeastolate+0.92 g/L Sodium Butyrate) was added, andculture temperature was changed to 35.5° C. The addition of the feedmedia was observed to dramatically enhance the production of solublerHuPH20 in the final stages of production. The reactor was harvested at14 days or when the viability of the cells dropped below 40%. Theprocess resulted in a final productivity of 17,000 Units per ml with amaximal cell density of 12 million cells/mL. At harvest, the culture wassampled for mycoplasma, bioburden, endotoxin and viral in vitro and invivo, TEM for viral particles and enzyme activity.

The culture was pumped by a peristaltic pump through four Millistakfiltration system modules (Millipore) in parallel, each containing alayer of diatomaceous earth graded to 4-8 μm and a layer of diatomaceousearth graded to 1.4-1.1 μm, followed by a cellulose membrane, thenthrough a second single Millistak filtration system (Millipore)containing a layer of diatomaceous earth graded to 0.4-0.11 μm and alayer of diatomaceous earth graded to <0.1 μm, followed by a cellulosemembrane, and then through a 0.22 μm final filter into a sterile singleuse flexible bag with a 350 L capacity. The harvested cell culture fluidwas supplemented with 10 mM EDTA and 10 mM Tris to a pH of 7.5. Theculture was concentrated 10× with a model tangential flow filtration(TFF) apparatus using four Sartoslice TFF 30 kDa molecular weightcut-off (MWCO) polyether sulfone (PES) filters (Sartorius), followed bya 10× buffer exchange with 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 into a 0.22μm final filter into a 50 L sterile storage bag.

The concentrated, diafiltered harvest was inactivated for virus. Priorto viral inactivation, a solution of 10% Triton X-100, 3% tri (n-butyl)phosphate (TNBP) was prepared. The concentrated, diafiltered harvest wasexposed to 1% Triton X-100, 0.3% TNBP for 1 hour in a 36 L glassreaction vessel immediately prior to purification on the Q column.

B. Purification of Gen2 sHuPH20

A Q Sepharose (Pharmacia) ion exchange column (9 L resin, H=29 cm, D=20cm) was prepared. Wash samples were collected for a determination of pH,conductivity and endotoxin (LAL) assay. The column was equilibrated with5 column volumes of 10 mM Tris, 20 mM Na₂SO4, pH 7.5. Following viralinactivation, the concentrated, diafiltered harvest was loaded onto theQ column at a flow rate of 100 cm/hr. The column was washed with 5column volumes of 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 and 10 mM Hepes, 50mM NaCl, pH 7.0. The protein was eluted with 10 mM Hepes, 400 mM NaCl,pH 7.0 into a 0.22 μm final filter into sterile bag. The eluate samplewas tested for bioburden, protein concentration and enzyme activity.A₂₈₀ absorbance reading were taken at the beginning and end of theexchange.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography wasnext performed. A Phenyl-Sepharose (PS) column (19-21 L resin, H=29 cm,D=30 cm) was prepared. The wash was collected and sampled for pH,conductivity and endotoxin (LAL assay). The column was equilibrated with5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate,0.1 mM CaCl₂, pH 7.0. The protein eluate from above was supplementedwith 2M ammonium sulfate, 1 M potassium phosphate and 1 M CaCl₂ stocksolutions to yield final concentrations of 5 mM, 0.5 M and 0.1 mM,respectively. The protein was loaded onto the PS column at a flow rateof 100 cm/hr. 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1mM CaCl₂ pH 7.0 was added at 100 cm/hr. The flow through was passedthrough a 0.22 μm final filter into a sterile bag. The flow through wassampled for bioburden, protein concentration and enzyme activity.

An aminophenyl boronate column (ProMedics; 21 L resin, H=29 cm, D=30 cm)was prepared. The wash was collected and sampled for pH, conductivityand endotoxin (LAL assay). The column was equilibrated with 5 columnvolumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate. The PSpurified protein was loaded onto the aminophenyl boronate column at aflow rate of 100 cm/hr. The column was washed with 5 mM potassiumphosphate, 0.5 M ammonium sulfate, pH 7.0. The column was washed with 20mM bicine, 0.5 M ammonium sulfate, pH 9.0. The column was washed with 20mM bicine, 100 mM sodium chloride, pH 9.0. The protein was eluted with50 mM Hepes, 100 mM NaCl, pH 6.9 and passed through a sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The hydroxyapatite (HAP) column (BioRad; 13 L resin, H=20 cm, D=30 cm)was prepared. The wash was collected and test for pH, conductivity andendotoxin (LAL assay). The column was equilibrated with 5 mM potassiumphosphate, 100 mM NaCl, 0.1 mM CaCl₂, pH 7.0. The aminophenyl boronatepurified protein was supplemented to final concentrations of 5 mMpotassium phosphate and 0.1 mM CaCl₂ and loaded onto the HAP column at aflow rate of 100 cm/hr. The column was washed with 5 mM potassiumphosphate, pH 7, 100 mM NaCl, 0.1 mM CaCl₂. The column was next washedwith 10 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1 mM CaCl₂. Theprotein was eluted with 70 mM potassium phosphate, pH 7.0 and passedthrough a 0.22 μm sterile filter into a sterile bag. The eluted samplewas tested for bioburden, protein concentration and enzyme activity.

The HAP purified protein was then passed through a viral removal filter.The sterilized Virosart filter (Sartorius) was first prepared by washingwith 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the filteredbuffer was sampled for pH and conductivity. The HAP purified protein waspumped via a peristaltic pump through the 20 nM viral removal filter.The filtered protein in 70 mM potassium phosphate, pH 7.0 was passedthrough a 0.22 μm final filter into a sterile bag. The viral filteredsample was tested for protein concentration, enzyme activity,oligosaccharide, monosaccharide and sialic acid profiling (as describedin Examples 9 to 10, below).

The protein in the filtrate was then concentrated to 10 mg/mL using a 10kD molecular weight cut off (MWCO) Sartocon Slice tangential flowfiltration (TFF) system (Sartorius). The filter was first prepared bywashing with 10 mM histidine, 130 mM NaCl, pH 6.0 and the permeate wassampled for pH and conductivity. Following concentration, theconcentrated protein was sampled and tested for protein concentrationand enzyme activity. A 6× buffer exchange was performed on theconcentrated protein into the final buffer: 10 mM histidine, 130 mMNaCl, pH 6.0. Following buffer exchange, the concentrated protein waspassed though a 0.22 μm filter into a 20 L sterile storage bag. Theprotein was sampled and tested for protein concentration, enzymeactivity, free sulfhydryl groups, oligosaccharide profiling andosmolarity (as described in Examples 9 to 10, below).

The sterile filtered bulk protein was then asceptically dispensed at 20mL into 30 mL sterile Teflon vials (Nalgene). The vials were then flashfrozen and stored at −20±5° C. Production and purification of solublerHuPH20 using this method yielded approximately 11 and 15 grams, withspecific activity of 95,000 units/mg to 120,000 units/mg.

C. Comparison of Production and Purification of Gen1 and Gen2 sHuPH20

The production and purification of Gen2 soluble rHuPH20 in a 300 Lbioreactor cell culture contained some changes in the protocols comparedto the production and purification Gen1 soluble rHuPH20 in a 100 Lbioreactor cell culture (described in Example 4). Table 25 sets forthexemplary differences, in addition to simple scale up changes, betweenthe methods.

TABLE 25 Exemplary differences between Gen1 and Gen2 soluble rHuPH20production and purification using the 100 L and 300 L bioreactor cellculture methods Process Difference Gen1 soluble rHuPH20 Gen2 solublerHuPH20 Cell line 3D35M 2B2 Media used to expand cell Contains 0.10 μMContains 20 μM inoculum methotrexate (0.045 mg/L) methotrexate (9 mg/L)Media in 6 L cultures Contains 0.10 μM Contains no methotrexate onwardsmethotrexate 36 L spinner flask No instrumentation Equipped with 20 Loperating volume. instrumentation that monitors and controls pH,dissolved oxygen, sparge and overlay gas flow rate. 32 L operatingvolume Final operating volume in Approx. 100 L in a 125 L Approx. 300 Lin a 400 L bioreactor bioreactor bioreactor (initial culture (initialculture volume + volume + 260 L) 65 L) Culture media in final NorHuInsulin 5.0 mg/L rHuInsulin bioreactor Media feed volume Scaled at 4%of the Scaled at 4% of the bioreactor cell culture bioreactor cellculture volume i.e. 3.4, 3.5 and 3.7 L, volume i.e. 10.4, 10.8,resulting in a target 11.2 and 11.7 L, resulting bioreactor volume of~92 L. in a target bioreactor volume of ~303 L. Media feed Feed #1Medium: CD Feed #1 Medium: 4x CD CHO + 50 g/L Glucose + 8 mM CHO + 33g/L Glucose + GlutaMAX ™-I 32 mM GlutaMAX ™-I + Feed #2 (CD CHO + 50 g/L16.6 g/L Yeastolate + 33 mg/L Glucose + 8 mM rHuInsulin GlutaMAX ™-I +1.1 g/L Feed #2: 2x CD CHO + 33 g/L Sodium Butyrate Glucose + 16 mM Feed#3: CD CHO + 50 g/L GlutaMAX ™-I + 33.4 g/L Glucose + 8 mM Yeastolate +0.92 g/L GlutaMAX ™-I + 1.1 g/L Sodium Butyrate Sodium Butyrate Feed #3:1x CD CHO + 50 g/L Glucose + 10 mM GlutaMAX ™-I + 50 g/L Yeastolate +1.80 g/L Sodium Butyrate Feed #4: 1x CD CHO + 33 g/L Glucose + 6.6 mMGlutaMAX ™-I + 50 g/L Yeastolate + 0.92 g/L Sodium Butyrate Filtrationof bioreactor cell Four polyethersulfone 1^(st) stage - Four modules inculture filters (8.0 μm, 0.65 μm, parallel, each with a layer 0.22 μmand 0.22 μm) in of diatomaceous earth series graded to 4-8 μm and a 100L storage bag layer of diatomaceous earth graded to 1.4-1.1 μm, followedby a cellulose membrane. 2^(nd) stage -single module containing a layerof diatomaceous earth graded to 0.4-0.11 μm and a layer of diatomaceousearth graded to <0.1 μm, followed by a cellulose membrane. 3^(rd)stage - 0.22 μm polyethersulfone filter 300 L storage bag Harvested cellculture is supplemented with 10 mM EDTA, 10 mM Tris to a pH of 7.5.Concentration and buffer Concentrate with 2 TFF Concentrate using fourexchange prior to with Millipore Spiral Sartorius Sartoslice TFFchromatography Polyethersulfone 30K 30K MWCO Filter MWCO Filter BufferExchange the Buffer Exchange the Concentrate 10× with 10 mM Concentrate6× with 10 mM Tris, 20 mM Na2SO4, Hepes, 25 mM NaCl, pH 7.5 pH 7.0 50 Lsterile storage bag 20 L sterile storage bag Viral inactivation prior toNone Viral inactivation chromatography performed with the addition of a1% Triton X- 100, 0.3% Tributyl Phosphate, pH 7.5, 1^(st) purificationstep (Q No absorbance reading A280 measurements at the sepharose)beginning and end Viral filtration after Pall DV-20 filter (20 nm)Sartorius Virosart filter (20 nm) chromatography Concentration andbuffer Hepes/saline pH 7.0 buffer Histidine/saline, pH 6.0 exchangeafter Protein concentrated to 1 mg/ml buffer chromatography Proteinconcentrated to 10 mg/ml Vial filling 5 mL and 1 mL fill 20 mL fillvolumes volumes Stored at ≦20° C. Stored at −≦30° C. Teflon/screw capGlass/rubber stopper Soluble rHuPH20 yield Approx. 400-700 mg Approx.11-25 g

Example 9 Determination of Enzymatic Activity of Soluble rHuPH20

Enzymatic activity of soluble rHuPH20 in samples such as cell cultures,purification fractions and purified solutions was determined using aturbidometric assay, which based on the formation of an insolubleprecipitate when hyaluronic acid binds with serum albumin. The activityis measured by incubating soluble rHuPH20 with sodium hyaluronate(hyaluronic acid) for a set period of time (10 minutes) and thenprecipitating the undigested sodium hyaluronate with the addition ofacidified serum albumin. The turbidity of the resulting sample ismeasured at 640 nm after a 30 minute development period. The decrease inturbidity resulting from enzyme activity on the sodium hyaluronatesubstrate is a measure of the soluble rHuPH20 enzymatic activity. Themethod is run using a calibration curve generated with dilutions of asoluble rHuPH20 assay working reference standard, and sample activitymeasurements are made relative to this calibration curve.

Dilutions of the sample were prepared in Enzyme Diluent Solutions. TheEnzyme Diluent Solution was prepared by dissolving 33.0±0.05 mg ofhydrolyzed gelatin in 25.0 mL of the 50 mM PIPES Reaction Buffer (140 mMNaCl, 50 mM PIPES, pH 5.5) and 25.0 mL of SWFI, and diluting 0.2 mL of25% Buminate solution into the mixture and vortexing for 30 seconds.This was performed within 2 hours of use and stored on ice until needed.The samples were diluted to an estimated 1-2 U/mL. Generally, themaximum dilution per step did not exceed 1:100 and the initial samplesize for the first dilution was not be less than 20 μL. The minimumsample volumes needed to perform the assay were: In-process Samples,FPLC Fractions: 80 μL; Tissue Culture Supernatants:1 mL; ConcentratedMaterial 80 μL; Purified or Final Step Material: 80 μL. The dilutionswere made in triplicate in a Low Protein Binding 96-well plate, and 30μL of each dilution was transferred to Optilux black/clear bottom plates(BD BioSciences).

Dilutions of known soluble rHuPH20 with a concentration of 2.5 U/mL wereprepared in Enzyme Diluent Solution to generate a standard curve andadded to the Optilux plate in triplicate. The dilutions included 0 U/mL,0.25 U/mL, 0.5 U/mL, 1.0 U/mL, 1.5 U/mL, 2.0 U/mL, and 2.5 U/mL.“Reagent blank” wells that contained 60 μL of Enzyme Diluent Solutionwere included in the plate as a negative control. The plate was thencovered and warmed on a heat block for 5 minutes at 37° C. The cover wasremoved and the plate was shaken for 10 seconds. After shaking, theplate was returned to the plate to the heat block and the MULTIDROP 384Liquid Handling Device was primed with the warm 0.25 mg/mL sodiumhyaluronate solution (prepared by dissolving 100 mg of sodiumhyaluronate (LifeCore Biomedical) in 20.0 mL of SWFI. This was mixed bygently rotating and/or rocking at 2-8° C. for 2-4 hours, or untilcompletely dissolved). The reaction plate was transferred to theMULTIDROP 384 and the reaction was initiated by pressing the start keyto dispense 30 μL sodium hyaluronate into each well. The plate was thenremoved from the MULTIDROP 384 and shaken for 10 seconds before beingtransferred to a heat block with the plate cover replaced. The plate wasincubated at 37° C. for 10 minutes

The MULTIDROP 384 was prepared to stop the reaction by priming themachine with Serum Working Solution and changing the volume setting to240 μL. (25 mL of Serum Stock Solution [1 volume of Horse Serum (Sigma)was diluted with 9 volumes of 500 mM Acetate Buffer Solution and the pHwas adjusted to 3.1 with hydrochloric acid] in 75 mL of 500 mM AcetateBuffer Solution). The plate was removed from the heat block and placedonto the MULTIDROP 384 and 240 μL of serum Working Solutions wasdispensed into the wells. The plate was removed and shaken on a platereader for 10 seconds. After a further 15 minutes, the turbidity of thesamples was measured at 640 nm and the enzyme activity (in U/mL) of eachsample was determined by fitting to the standard curve.

Specific activity (Units/mg) was calculated by dividing the enzymeactivity (U/ml) by the protein concentration (mg/mL).

Example 10 Determination of Sialic Acid and Monosaccharide Content

The sialic acid and monosaccharide content of soluble rHuPH20 can beassessed by reverse phase liquid chromatography (RPLC) followinghydrolysis with trifluoroacetic acid. In one example, the sialic acidand monosaccharide content of purified hyaluronidase lot # HUB0701E (1.2mg/mL; produced and purified essentially as described in Example 8) wasdetermined. Briefly, 100 μg sample was hydrolyzed with 40% (v/v)trifluoroacetic acid at 100° C. for 4 hours in duplicate.

Following hydrolysis, the samples were dried down and resuspended in 300μL water. A 45 μL aliquot from each re-suspended sample was transferredto a new tube and dried down, and 10 μL of a 10 mg/mL sodium acetatesolution was added to each. The released monosaccharides werefluorescently labeled by the addition of 50 μL of a solution containing30 mg/mL 2-aminobenzoic acid, 20 mg/mL sodium cyanoborohydride,approximately 40 mg/mL sodium acetate and 20 mg/mL boric acid inmethanol. The mixture was incubated for 30 minutes at 80° C. in thedark. The derivitization reaction was quenched by the addition of 440 μLof mobile phase A (0.2% (v/v) n-butylamine, 0.5% (v/v) phosphoric acid,1% (v/v) tetrahydrofuran). A matrix blank of water also was hydrolyzedand derivitized as described for the hyaluronidase sample as a negativecontrol. The released monosaccharides were separated by RPLC using anOctadecyl (C₁₈) reverse phase column (4.6×250 mm, 5 μm particle size; J.T. Baker) and monitored by fluorescence detection (360 nm excitation,425 nm emission). Quantitation of the monosaccharide content was made bycomparison of the chromatograms from the hyaluronidase sample withchromatograms of monosaccharide standards including N-D-glucosamine(GlcN), N-D-galactosamine (GaiN), galactose, fucose and mannose. Table26 presents the molar ratio of each monosaccharide per hyaluronidasemolecule.

TABLE 26 Monosaccharide content of soluble rHuPH20 Lot Replicate GlcNGalN Galactose Mannose Fucose HUB0701E 1 14.28 0.07* 6.19 25.28 2.69 213.66 0.08* 6.00 24.34 2.61 Average 13.97 0.08* 6.10 24.81 2.65 *GalNresults were below the limit of detection

Example 11 C-terminal Heterogeneity of Soluble rHuPH20 from 3D35M and2B2 Cells

C-terminal sequencing was performed on two lots of sHuPH20 produced andpurified from 3D35M cells in a 100 L bioreactor volume (Lot HUA0505MA)and 2B2 cells in a 300 L bioreactor volume (Lot HUB0701EB). The lotswere separately digested with endoproteinase Asp-N, which specificallycleaves peptide bonds N-terminally at aspartic and cysteic acid. Thisreleases the C-terminal portion of the soluble rHuPH20 at the asparticacid at position 431 of SEQ ID NO:4. The C-terminal fragments wereseparated and characterized to determine the sequence and abundance ofeach population in Lot HUA0505MA and Lot HUB0701EB.

It was observed that the soluble rHuPH20 preparations from 3D35M cellsand 2B2 cells displayed heterogeneity, and contained polypeptides thatdiffered from one another in their C-terminal sequence (Tables 27 and28). This heterogeneity is likely the result of C-terminal cleavage ofthe expressed 447 amino acid polypeptide (SEQ ID NO:4) by peptidasespresent in the cell culture medium or other solutions during theproduction and purification process. The polypeptides in the solublerHuPH20 preparations have amino acid sequences corresponding to aminoacids 1-447, 1-446, 1-445, 1-444 and 1-443 of the soluble rHuPH20sequence set forth SEQ ID NO:4. The full amino acid sequence of each ofthese polypeptides is forth in SEQ ID NOS: 4 to 8, respectively. Asnoted in tables 27 and 28, the abundance of each polypeptide in thesoluble rHuPH20 preparations from 3D35M cells and 2B2 cells differs.

TABLE 27 Analysis of C-terminal fragments from Lot HUA0505MA Amino acidposition (relative Frag- to SEQ Theor. Exp. Elution ment ID NO: 4)Sequence Mass Mass Error time Abundance D28a 431-447 DAFKLPPMETEEPQIFY2053.97 2054.42 0.45 99.87 0.2% (SEQ ID NO: 66) D28b 431-446DAFKLPPMETEEPQIF 1890.91 1891.28 0.37 97.02 18.4% (SEQ ID NO: 67) D28c431-445 DAFKLPPMETEEPQI 1743.84 1744.17 0.33 86.4 11.8% (SEQ ID NO: 68)D28d 431-444 DAFKLPPMETEEPQ 1630.70 1631.07 0.32 74.15 56.1%(SEQ ID NO: 69) D28e 431-443 DAFKLPPMETEEP 1502.70 1502.98 0.28 77.3613.6% (SEQ ID NO: 70) D28f 431-442 DAFKLPPMETEE 1405.64 ND N/A N/A 0.0%(SEQ ID NO: 71)

TABLE 28 Analysis of C-terminal fragments from Lot HUB0701EB Amino acidposition (relative Frag- to SEQ Theor. Exp. Elution ment ID NO: 4)Sequence Mass Mass Error time Abundance D28a 431-477 DAFKLPPMETEEPQIFY2053.97 2054.42 0.45 99.89 1.9% (SEQ ID NO: 66) D28b 431-446DAFKLPPMETEEPQIF 1890.91 1891.36 0.45 96.92 46.7% (SEQ ID NO: 67) D28c431-445 DAFKLPPMETEEPQI 1743.84 1744.24 0.40 85.98 16.7% (SEQ ID NO: 68)D28d 431-444 DAFKLPPMETEEPQ 1630.70 1631.14 0.39 73.9 27.8%(SEQ ID NO: 69) D28e 431-443 DAFKLPPMETEEP 1502.70 1503.03 0.33 77.026.9% (SEQ ID NO: 70) D28f 431-442 DAFKLPPMETEE 1405.64 ND N/A N/A 0.0%(SEQ ID NO: 71)

Example 12 Production and Purification of Soluble rHuPH20 in 2500 LBioreactor Cell Culture

The production and purification of soluble rHuPH20 can be scaled up froma 300 L batch-fed bioreactor process (described in Example 8) to a 2500L batch-fed bioreactor process. Like production of rHuPH20 in a 300 Lbioreactor cell culture, the production of rHuPH20 in a 2500 Lbioreactor cell culture is performed by first thawing and expanding avial of 2B2 cells, culturing in a bioreactor, harvesting and clarifyingthe culture, concentrating and buffer-exchanging the harvest, followedby viral inactivation. The rHuPH20 is then purified from the concentrateusing a series of purification steps that utilize Q sepharose, Phenylsepharose, aminophenyl boronate and hydroxyapatite boronate, followed byviral filtration.

1. Cell Culture Expansion

To generate higher cell numbers required for seeding the 2500 Lbioreactor cell culture compared to the 300 L culture, the cell cultureis serially expanded through a 125 mL shaker flask, a 250 mL shaker, a 1L shaker flask, two 2 L shaker flasks, six 2 L shaker flasks, a 25 LWAVE Bioreactor™ (GE Healthcare Life Sciences), a 100 L WAVEBioreactor™, and a 600 L stirred tank seed bioreactor (ABEC, Inc.Bethlehem, Pa.; Stainless Technology division). At each expansion, thetarget seeding density is 4×10⁵ cells/mL The temperature throughout theexpansion is 37° C. (or between 36° C. and 38° C.) with 7% CO₂ (orbetween 6-8% CO₂). The flasks are agitated at approximately 110 RPM (or90-130 RPM), the 25 L and 100 L WAVE Bioreactor™ are rocked at 20 RPM(or 15-25 or 18-22 RPM, respectively) and the 600 L seed bioreactor isagitated at 90 RPM (or 85-95 RPM).

First, a vial of 2B2 cells (1×10⁷ cells) from the working cell bank isthawed in a 37° C. water bath for approximately 2 minutes (preferably nomore than 5 minutes) before media is added and the cells arecentrifuged. The cells are re-suspended to approximately 25 mL (orbetween 20-30 mL) with fresh media (CD CHO AGT™ with 40 mL/L (8 mM)GlutaMAX™-I and 20 μM methotrexate in a 125 mL shaker flask and placedin a 37° C., 7% CO₂ incubator. When the cell density reachesapproximately 8×10⁵ cells/mL, the culture is transferred into a 250 mLshake flask in a 50 mL culture volume (or 45-55 mL). Followingincubation, when the cell density reaches approximately 1.6×10⁶cells/mL, the culture is expanded into a 1 L flask in 200 mL culturevolume (or 190-210 mL) and incubated. When the cell density in the 1 Lflask reaches approximately 1.6×10⁶ cells/mL, the culture is expandedinto 2×2 L flasks, each with a total culture volume of approximately 400mL (or between 350-450 mL per flask), and incubated. When the celldensity in the 2 L flasks reaches approximately 1.2×10⁶ cells/mL, theculture is expanded into 6×2 L flasks, each with a total culture volumeof approximately 400 mL (or between 350-450 mL per flask), andincubated. When the cell density in the 2 L flasks reaches approximately2.5×10⁶ cells/mL, the culture is expanded into a 25 L WAVE Bioreactor™,with a total culture volume of approximately 15 L (or between 14-16 L)and incubated with an air flow of 1.5 L/minute.

When the cell density in the 25 L WAVE Bioreactor™ reaches approximately2.2×10⁶ cells/mL, the culture is expanded into a 100 L WAVE Bioreactor™,with a total culture volume of approximately 80 L (or between 75-85 L),using CD-CHO AGT™ media that is supplemented with 3.6 g/L methotrexate,40 mL/L GlutaMAX™-I and 1 mL/L 1N NaOH, and incubated with an air flowof 1.5 L/minute. When the cell density in the 100 L WAVE Bioreactor™reaches approximately 2.6×10⁶ cells/mL, the culture is expanded into a600 L seed bioreactor ABEC, Inc. Bethlehem, Pa.; Stainless Technologydivision) with a total culture volume of approximately 480 L (or between440-520 L) using CD-CHO AGT™ media that is supplemented with 40 mL/LGlutaMAX™-I and incubated until the cell density in the 600 L bioreactorreaches approximately 1.6×10⁶ cells/mL.

2. rHuPH20 Production

A 3500 L bioreactor with a total volume of 3523 L and a working volumeof 500-2500 L (ABEC, Inc, Bethlehem, Pa.) is used for high yieldproduction of rHuPH20. Following sterilization, approximately 1800-2000L CD-CHO AGT™ media containing 24.3 g/L powdered CD-CHO AGT™,supplemented with 40 mL/L GlutaMAX™-I and 5 mg/L rHulnsulin is added tothe bioreactor. Parameters are set to: temperature setpoint, 37° C.;Impeller Speed 75 RPM; Vessel Pressure: 5 psi; Air Sparge 18 L/min;dissolved oxygen: 25%; pH≦7.2. Before use, the reactor is checked forcontamination. Approximately between 300-500 L of cells (depending oncell count) from the 600 L seed bioreactor culture are inoculated intothe cell culture medium in the 3500 L bioreactor at an inoculationdensity of 4.0×10⁵ viable cells per ml, to reach a total volume of 2100L. During the 14 day cell incubation, the bioreactor is sampled dailyfor cell viability, cell density, pH verification, and enzymaticactivity. Temperature and dissolved oxygen also are monitored closely.

Nutrient feeds are added during the 14 day bioreactor run, each atapproximately 4% v/v. At day 5, approximately 84 L (or 4% v/v) of Feed#1 Medium (81 g/L powdered CD-CHO AGT™+33 g/L Glucose+13.3 mL/LGlutaMAX™-I+83.3 g/L Yeastolate+33 mg/L rHuInsulin) is added. At day 7,approximately 87 L (or 4% v/v) of Feed #2 (40.5 g/L powdered CD-CHOAGT™+33 g/L Glucose+66.7 mL/L GlutaMAX™-I+166.7 g/L Yeastolate+0.92 g/LSodium butyrate) is added, and culture temperature is changed to 36.5°C. At day 9, approximately 91 L (or 4% v/v) of Feed #3 (20.3 g/Lpowdered CD-CHO AGT™+50 g/L Glucose+50 mL/L GlutaMAX™-I+250 g/LYeastolate+1.8 g/L Sodium butyrate) is added, and culture temperature ischanged to 36° C. At day 11, approximately 94 L (or 4% v/v) of Feed #4(20.3 g/L powdered CD-CHO AGT™+33.3 g/L Glucose+33.3 mL/LGlutaMAX™-I+250 g/L Yeastolate+0.92 g/L Sodium butyrate) is added, andculture temperature is changed to 35.5° C. The reactor is harvested at14 days, yielding 2400-2600 L harvest (typically approximately 2500 L).

The culture is pressure transferred through 20 Millistak filtrationsystem modules (Millipore) in parallel, each containing a layer ofdiatomaceous earth graded to 4-8 μm and a layer of diatomaceous earthgraded to 1.4-1.1 μm, followed by a cellulose membrane, then through asecond Millistak filtration system (Millipore) containing 10 modules,each with a layer of diatomaceous earth graded to 0.4-0.11 μm and alayer of diatomaceous earth graded to <0.1 μm, followed by a cellulosemembrane, and then through a 0.22 μm final filter into a sterile singleuse flexible bag with a 350 L capacity. The harvested cell culture fluidis supplemented with 10 mM EDTA and 10 mM Tris, pH 8.4, to a target pHof 7.5. The culture is concentrated 1 ox with a tangential flowfiltration (TFF) apparatus (Pall) using 18-21 m² of Sartoslice TFF 30kDa molecular weight cut-off (MWCO) polyether sulfone (PES) filters(Sartorius), followed by a 10× buffer exchange with 10 mM Tris, 20 mMNa₂SO₄, pH 7.5 into a 0.22 μm final filter into a 350 L sterile storagebag.

The concentrated, diafiltered harvest is inactivated for virus. Prior toviral inactivation, a solution of 10% Triton X-100, 3% tri (n-butyl)phosphate (TNBP) was prepared. The concentrated, diafiltered harvest wasexposed to 1% Triton X-100, 0.3% TNBP up to 2 hours in 500 L stainlesssteel reaction vessels immediately prior to purification on the Qcolumn.

B. Purification of Gen2 rHuPH20

A Q Sepharose (Pharmacia) ion exchange column (81 L resin, H=26 cm, D=63cm) is prepared. The column is equilibrated with 5 column volumes of 10mM Tris, 20 mM Na₂SO₄, pH 7.5. Following viral inactivation, theconcentrated, diafiltered, viral-inactivated harvest of approximately250 L is loaded onto the Q column at a flow rate of 150 cm/hr. Thecolumn is washed with 5 column volumes of 10 mM Tris, 20 mM Na₂SO₄, pH7.5 and 10 mM Hepes, 50 mM NaCl, pH 7.0. The protein is eluted with 10mM Hepes, 400 mM NaCl, pH 7.0 into a 0.22 μm final filter into sterilebag. The eluate sample is tested for bioburden, protein concentrationand enzyme activity. A₂₈₀ absorbance reading were taken at the beginningand end of the exchange.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography isnext performed. A Phenyl-Sepharose (PS) column (176 L resin, H=35 cm,D=80 cm) is prepared. The column is equilibrated with 5 column volumesof 5 mM potassium phosphate, 0.5 M ammonium sulfate, 0.1 mM CaCl₂, pH7.0. The protein eluate from above is supplemented with 2M ammoniumsulfate, 1 M potassium phosphate and 1 M CaCl₂ stock solutions to yieldfinal concentrations of 5 mM, 0.5 M and 0.1 mM, respectively. Theprotein is loaded onto the PS column at a flow rate of 100 cm/hr. 5 mMpotassium phosphate, 0.5 M ammonium sulfate and 0.1 mM CaCl₂ pH 7.0 wasadded at 100 cm/hr. The flow through is passed through a 0.22 μm finalfilter into a sterile bag. The flow through is sampled for bioburden,protein concentration and enzyme activity.

An aminophenyl boronate column (ProMedics; 176 L resin, H=35 cm, D=80cm) is then prepared. The column is equilibrated with 5 column volumesof 5 mM potassium phosphate, 0.5 M ammonium sulfate. The PS-purifiedprotein is loaded onto the aminophenyl boronate column at a flow rate of50 cm/hr. The flow rate was increased to 100 cm/hr for the remainder ofthe process. The column was first washed with 5 mM potassium phosphate,0.5 M ammonium sulfate, pH 7.0, then 20 mM bicine, 0.5 M ammoniumsulfate, pH 9.0, and then with 20 mM bicine, 100 mM sodium chloride, pH9.0. The protein is eluted with 50 mM Hepes, 100 mM NaCl, pH 6.9 andpassed through a sterile filter into a sterile bag. The eluted sample istested for bioburden, protein concentration and enzyme activity.

The hydroxyapatite (HAP) column (BioRad; 116 L resin, H=23 cm, D=80 cm)is prepared. The column is equilibrated with 5 mM potassium phosphate,100 mM NaCl, 0.1 mM CaCl₂, pH 7.0. The aminophenyl boronate purifiedprotein is supplemented to final concentrations of 5 mM potassiumphosphate and 0.1 mM CaCl₂ and loaded onto the HAP column at a flow rateof 100 cm/hr. The column is first washed with 5 mM potassium phosphate,pH 7, 100 mM NaCl, 0.1 mM CaCl₂, then 10 mM potassium phosphate, pH7;100 mM NaCl, 0.1 mM CaCl₂. The protein is eluted with 70 mM potassiumphosphate, pH 7.0 and passed through a 0.22 μm sterile filter into asterile bag. The eluted sample is tested for bioburden, proteinconcentration and enzyme activity.

The HAP purified protein is then passed through a viral removal filter.The sterilized Viosart filter (Sartorius) is first prepared by washingwith 2 L of 70 mM potassium phosphate, pH 7.0. The HAP purified proteinis pumped via a peristaltic pump through the 20 nM viral removal filter.The filtered protein in 70 mM potassium phosphate, pH 7.0 is then passedthrough a 0.22 μm final filter into a sterile bag. The viral filteredsample is tested for protein concentration, enzyme activity,oligosaccharide, monosaccharide and sialic acid profiling (as describedin Examples 9 to 10, below).

The protein in the filtrate was then concentrated 8-12× using three 10kD molecular weight cut off (MWCO) Sartocon PES cassettes, each with afilter surface area of 0.7 m², for a total surface area of 2.1 m².Following concentration, the concentrated protein was sampled and testedfor protein concentration and enzyme activity. A 10× diafiltration isthen performed on the concentrated protein. This can be performed one oftwo ways: 1) using a 20 mM histidine, 130 mM NaCl, pH 6.5 buffer and 1%polysorbate 80; or 2) using a 10 mM histidine, 130 mM NaCl, pH 6.5buffer. The concentrated, diafiltered bulk protein is at a concentrationof approximately 10 mg/mL. Following buffer exchange, the concentratedprotein is passed though a 0.22 μm filter into a 20 L sterile storagebag.

The sterile filtered bulk protein is then asceptically dispensed at 400mL into 1 L sterile PFA Nalgene bottles. The bottles are then flashfrozen in a liquid nitrogen bath and stored at less that −20° C. for thebulk protein that does not contain the polysorbate 80, and less that−70° C. for the bulk protein that does contain the polysorbate 80.

Table 29 sets forth some exemplary differences between production ofrHuPH20 in a 300 L and 2500 L bioreactor culture

TABLE 29 Exemplary differences between production of rHuPH20 in a 300 Land 2500 L bioreactor culture Process Difference 300 L cell culture 2500L cell culture Cell line 2B2 2B2 Media used to expand cell Contains 20μM Contains 20 μM inoculum methotrexate (9 mg/L) methotrexate (9 mg/L)Cell expansion Expanded through 125 mL Expanded through 125 mL flask,250 mL flask, 1 L flask, 250 mL flask, 1 L flask, 6 L flask and 36 Lflask, 2 × 2 L shaker flask. flasks, 6 × 2 L shaker flasks, 25 L WAVEBioreactor ™, 100 L WAVE Bioreactor ™, and a 600 L stirred tank seedbioreactor Final operating volume in Approx. 300 L in a 400 L Approx.2500 L in a 3500 L bioreactor bioreactor bioreactor Culture media in atCD CHO with 5.0 mg/L CD CHO AGT ™ with 5.0 mg/L bioreactor inoculationrHuInsulin rHuInsulin 40 mL/L (8 mM) 40 mL/L (8 mM) GlutaMAX ™-IGlutaMAX ™-I Media feed volume Scaled at 4% of the Scaled at 4% of thebioreactor cell culture bioreactor cell culture volume i.e.approximately volume i.e. approximately 10.4, 10.8, 11.2 and 11.7 L, 84,87, 91, and 94 L, resulting in a target resulting in a target bioreactorvolume of ~300 L. bioreactor volume of ~2500 L. Media feed Feed #1Medium: 4x CD Feed #1 Medium: 81 g/L CHO + 33 g/L Glucose + powderedCD-CHO 32 mM GlutaMAX ™-I + AGT ™ + 33 g/L Glucose + 16.6 g/LYeastolate + 33 mg/L 26.6 mM GlutaMAX ™-I + rHuInsulin 83.3 g/LYeastolate + 33 mg/L Feed #2: 2x CD CHO + 33 g/L rHuInsulin Glucose + 16mM Feed #2: 40.5 g/L GlutaMAX ™-I + 33.4 g/L powdered CD-CHOYeastolate + 0.92 g/L AGT ™ + 33 g/L Glucose + Sodium Butyrate 13.4 mMGlutaMAX ™-I + Feed #3: 1x CD CHO + 50 g/L 166.7 g/L Yeastolate +Glucose + 10 mM 0.92 g/L Sodium butyrate GlutaMAX ™-I + 50 g/L Feed #3:20.3 g/L Yeastolate + 1.80 g/L powdered CD-CHO Sodium Butyrate AGT ™ +50 g/L Glucose + Feed #4: 1x CD CHO + 33 g/L 10 mM GlutaMAX ™-I +Glucose + 6.7 mM 250 g/L Yeastolate + 1.8 g/L GlutaMAX ™-I + 50 g/LSodium butyrate Yeastolate + 0.92 g/L Feed #4: 20.3 g/L Sodium Butyratepowdered CD-CHO AGT ™ + 33.3 g/L Glucose + 6.7 mM GlutaMAX ™-I + 250 g/LYeastolate + 0.92 g/L Sodium butyrate Filtration of bioreactor cell1^(st) stage - 4 modules in 1 t stage - 20 modules in culture parallel,each with a layer parallel, each with a layer of diatomaceous earth ofdiatomaceous earth graded to 4-8 μm and a graded to 4-8 μm and a layerof diatomaceous layer of diatomaceous earth graded to 1.4-1.1 μm, earthgraded to 1.4-1.1 μm, followed by a cellulose followed by a cellulosemembrane. membrane. 2^(nd) stage -single module 2^(nd) stage -10 modulescontaining a layer of containing a layer of diatomaceous earth gradeddiatomaceous earth graded to 0.4-0.11 μm and a layer to 0.4-0.11 μm anda layer of diatomaceous earth of diatomaceous earth graded to <0.1 μm,graded to <0.1 μm, followed by a cellulose followed by a cellulosemembrane. membrane. 3^(rd) stage - 0.22 μm 3^(rd) stage - 0.22 μmpolyethersulfone filter polyethersulfone filter 300 L storage bag 350 Lstorage bag Concentration and buffer Concentrate using four Concentratewith 2 TFF exchange prior to Sartorius Sartoslice TFF with MilliporeSpiral chromatography 30K MWCO Filter Polyethersulfone 30K BufferExchange the MWCO Filter Concentrate 10× with 10 mM Buffer Exchange theTris, 20 mM Na2SO4, Concentrate 6× with 10 mM pH 7.5 Hepes, 25 mM NaCl,50 L sterile storage bag pH 7.0 20 L sterile storage bag Q sepharosecolumn 9 L resin, H = 29 cm, D = 81 L resin, H = 26 cm, D = 20 cm 63 cmA Phenyl-Sepharose (PS) 19-21 L resin, H = 29 cm, 176 L resin, H = 35cm, D = column D = 30 cm 80 cm aminophenyl boronate 21 L resin, H = 29cm, D = 176 L resin, H = 35 cm, D = column 30 cm 80 cm hydroxyapatite(HAP) 13 L resin, H = 20 cm, D = 116 L resin, H = 23 cm, D = column 30cm 80 cm Protein Concentration Single 10 kD MWCO Three 10 kD molecularSartocon Slice tangential weight cut off (MWCO) flow filtration (TFF)Sartocon PES cassettes 6× buffer exchange with 10x diafiltration with:10 mM histidine, 130 mM 1) 20 mM histidine, 130 mM NaCl, pH 6.0 NaCl, pH6.5 buffer and 1% polysorbate 80; or 2) 10 mM histidine, 130 mM NaCl, pH6.5 buffer. Vial filling 20 mL fill volumes 400 mL fill volumes Storedat ≦20° C. Stored at ≦20° C. if protein does not polysorbate 80, or ≦70°C. if protein does contain polysorbate 80

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A harvested cell culture fluid, comprising soluble rHuPH20 with anenzymatic activity that is greater than 5000 units/mL.
 2. The harvestedcell culture fluid of claim 1, wherein the enzymatic activity is or isabout 10,000, 12,000, 14,000, 16,000, 18,000, 20,000, 22,000 or 24,000units/mL.
 3. The harvested cell culture fluid of claim 1, wherein theculture medium is produced by a method comprising: a) inoculating cellmedium in a bioreactor with an inoculum of cells that encode solublerHuPH20 to produce a cell culture, wherein: the cells comprise between150 and 300 copies of nucleic acid encoding soluble rHuPH20; thebioreactor contains at least 100 liters of cell culture; and about10¹⁰-10¹¹ cells are inoculated per 100 liters cell culture; and thecells are cultured at a set temperature; b) feeding the cells with afirst feed medium containing glucose, L-alanyl-L-glutamine, humaninsulin and yeast extract in amounts sufficient to increase cell growthand peak cell density, and to increase soluble PH20 synthesis, whereinthe feed medium is added to the culture at a volume of 0.5% or about0.5% to 20% or about 20% of the cell culture volume; c) feeding thecells with a second feed medium containing glucose,L-alanyl-L-glutamine, yeast extract and sodium butyrate in amountssufficient to increase soluble rHuPH20 synthesis and induce cell cyclearrest; and lowering the temperature compared to the temperature in stepa) to a temperature sufficient to increase cell cycle arrest, increasecell viability and stabilize the soluble hyaluronidase; wherein: theamount of L-alanyl-L-glutamine is decreased compared to the amount ofL-alanyl-L-glutamine in step b); the amount of yeast extract isincreased compared to the amount of yeast extract in step b); and thefeed medium is added to the culture at a volume of 0.5% or about 0.5% to20% or about 20% of the cell culture volume; d) feeding the cells with athird feed medium containing glucose, L-alanyl-L-glutamine, yeastextract and sodium butyrate in amounts sufficient to increase solublerHuPH20 synthesis and increase cell cycle arrest, and lowering thetemperature compared to the temperature in step c) to a temperaturesufficient to increase cell cycle arrest, increase cell viability andstabilize the soluble hyaluronidase; wherein: the amount ofL-alanyl-L-glutamine is decreased compared to the amount ofL-alanyl-L-glutamine in step c); the amounts of yeast extract, glucoseand sodium butyrate are increased compared to the amounts of yeastextract, glucose and sodium butyrate in step c); and the feed medium isadded to the culture at a volume of 0.5% or about 0.5% to 20% or about20% of the cell culture volume; e) feeding the cells with a fourth feedmedium containing glucose, L-alanyl-L-glutamine, yeast extract andsodium butyrate in amounts sufficient to increase soluble rHuPH20synthesis and increase cell cycle arrest, and lowering the temperaturecompared to the temperature in step d) to a temperature sufficient toincrease cell cycle arrest, increase cell viability and stabilize thesoluble hyaluronidase; wherein: the amount of L-alanyl-L-glutamine andglucose is decreased compared to the amount of L-alanyl-L-glutamine andglucose in step d); the amount of sodium butyrate is decreased comparedto the amount of sodium butyrate in step d); and the feed medium isadded to the culture at a volume of 0.5% or about 0.5% to 20% or about20% of the cell culture volume; f) continuing to culture the cells untilviability drops below at least or about 50%; and g) harvesting the cellculture fluid.
 4. The harvested cell culture fluid of claim 3, whereinthe temperature in step a) is or is about 37° C.
 5. The harvested cellculture fluid of claim 3, wherein the temperature in step c) is or isabout 36.5° C.
 6. The harvested cell culture fluid of claim 3, whereinthe temperature in step d) is 36° C.
 7. The harvested cell culture fluidof claim 3, wherein the temperature in step e) is 35.5° C.
 8. Theharvested cell culture fluid of claim 3, wherein the harvest cellculture fluid is filtered.
 9. The harvested cell culture fluid of claim3, wherein a feed medium is added to the culture at a volume of 4% orabout 4% of the cell culture volume.
 10. The harvested cell culturefluid of claim 1, wherein the cell culture medium is produced by amethod, comprising: a) inoculating cell medium in a bioreactor with aninoculum of cells that encode soluble rHuPH20 to produce a cell culture,wherein: the cells comprise between 150 and 300 copies of nucleic acidencoding soluble rHuPH20; the bioreactor contains at least 100 liters ofcell culture; the inoculation cell density is at or about 4×10⁵cells/mL; and the cells are cultured at or at about 37° C.; b) feedingthe cells with a first feed medium containing or containing about 33 g/Lglucose, 32 mM L-alanyl-L-glutamine, 16.6 g/L yeast extract and 33 mg/Linsulin, wherein the feed medium is added to the culture at a volume of4% or about 4% of the cell culture volume; c) feeding the cells with asecond feed medium containing or containing about 33 g/L glucose, 16 mML-alanyl-L-glutamine, 33.4 g/L yeast extract and 0.92 g/L sodiumbutyrate, wherein the feed medium is added to the culture at a volume of4% or about 4% of the cell culture volume; and the temperature islowered to 36.5° C.; d) feeding the cells with a third feed mediumcontaining or containing about 50 g/L glucose, 10 mML-alanyl-L-glutamine, 50 g/L yeast extract and 1.8 g/L sodium butyrate,wherein the feed medium is added to the culture at a volume of 4% orabout 4% of the cell culture volume; and the temperature is lowered to36° C.; e) feeding the cells with a fourth feed medium containing orcontaining about 33 g/L glucose, 6.6 mM L-alanyl-L-glutamine, 50 g/Lyeast extract and 0.92 g/L sodium butyrate, wherein the feed medium isadded to the culture at a volume of 4% or about 4% of the cell culturevolume; and the temperature is lowered to 35.5° C.; f) continuing toculture the cells until viability drops below at least or about 50%; g)harvesting the cell culture fluid; and h) filtering the harvested cellculture fluid.
 11. The harvested cell culture fluid of claim 1, whereinthe cell culture medium is produced by a method, comprising: a)inoculating cell medium in a bioreactor with an inoculum of cells thatencode soluble rHuPH20 to produce a cell culture, wherein: the cellscomprise between 150 and 300 copies of nucleic acid encoding solublerHuPH20; the bioreactor contains at least 100 liters of cell culture;the inoculation cell density is at or about 4×10⁵ cells/mL; and thecells are cultured at or at about 37° C.; b) feeding the cells with afirst feed medium containing or containing about 33 g/L glucose, 32 mML-alanyl-L-glutamine, 83.3 g/L yeast extract and 33 mg/L insulin,wherein the feed medium is added to the culture at a volume of 4% orabout 4% of the cell culture volume; c) feeding the cells with a secondfeed medium containing or containing about 33 g/L glucose, 13 mML-alanyl-L-glutamine, 166.7 g/L yeast extract and 0.92 g/L sodiumbutyrate, wherein the feed medium is added to the culture at a volume of4% or about 4% of the cell culture volume; and the temperature islowered to 36.5° C.; d) feeding the cells with a third feed mediumcontaining or containing about 50 g/L glucose, 10 mML-alanyl-L-glutamine, 250 g/L yeast extract and 1.8 g/L sodium butyrate,wherein the feed medium is added to the culture at a volume of 4% orabout 4% of the cell culture volume; and the temperature is lowered to36° C.; e) feeding the cells with a fourth feed medium containing orcontaining about 33 g/L glucose, 6.7 mM L-alanyl-L-glutamine, 250 g/Lyeast extract and 0.92 g/L sodium butyrate, wherein the feed medium isadded to the culture at a volume of 4% or about 4% of the cell culturevolume; and the temperature is lowered to 35.5° C.; f) continuing toculture the cells until viability drops below at least or about 50%; g)harvesting the cell culture fluid; and h) filtering the harvested cellculture fluid.
 12. The harvested cell culture fluid of claim 1, whereinthe volume of cell culture in the bioreactor is or is about 200, 300,400, 500, 1000, 1500, 2000, 2500, 3000 or 3500 liters.
 13. The harvestedcell culture fluid of claim 3, wherein the cells that encode solublerHuPH20 are DG44 CHO cells.
 14. The harvested cell culture fluid ofclaim 10, wherein the cells that encode soluble rHuPH20 are DG44 CHOcells.
 15. The harvested cell culture fluid of claim 11, wherein thecells that encode soluble rHuPH20 are DG44 CHO cells.
 16. The harvestedcell culture fluid of claim 3, wherein the rHuPH20 is encoded by nucleicacid set forth in SEQ ID NO:47.
 17. The harvested cell culture fluid ofclaim 10, wherein the rHuPH20 is encoded by nucleic acid set forth inSEQ ID NO:47.
 18. The harvested cell culture fluid of claim 11, whereinthe rHuPH20 is encoded by nucleic acid set forth in SEQ ID NO:47.